Note: Descriptions are shown in the official language in which they were submitted.
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Use of peptidylglycine alpha-amidating monooxygenase (PAM) for therapeutic
purpose
The present invention is directed to the use of peptidylglycine alpha-
amidating monooxygenase (PAM)
as medicament.
State of the Art
Biologically active peptide hormones fulfill the function as signaling
molecules. Most bioactive peptide
hormones are synthesized from larger, inactive precursor peptides. During
their biosynthesis, those
peptides undergo several co- and posttranslational modifications, including
cleavage of signal peptides,
endoproteolytic cleavage of the precursor pro-peptides by specific
endopeptidases mostly at pairs of
basic residues, removal of basic residues by carboxypeptidases, formations of
disulfide bonds and N-
and 0-glyeosylation (Eipper et al. 1993. Protein Science 2(4): 489-97). More
than half of the known
neural and endocrine peptides require an additional modification step to gain
full biological activity
involving the formation of a c-terminal alpha-amide group (Guembe, et al.
1999. J Histochem Cytochem
47(5): 623 36). This final step of peptide hormone biosynthesis involves the
action of the bifunctional
enzyme peptidylglycine alpha-amidating monooxygenase (PAM). PAM specifically
recognizes c-
terminal glycine residues in its substrates, cleaves glyoxylate from the
peptide's c-terminal glycine
residue in a two-step enzymatic reaction leading to the formation of c-
terminally alpha-amidated peptide
hormones, wherein the resulting alpha-amide group originates from the cleaved
c-terminal glycine
(Prigge et al. 2004. Science 304(5672): 864-67). This amidation reaction takes
place in the lumen of
secretory granules prior to exocytosis of the amidated product (Martinez and
Treston 1996. Molecular
and Cellular Endocrinol 123: 113-17). Alpha-amidatcd peptides arc for example
adrenomedullin,
substance P. vasopressin, neuropeptide Y, Amylin, calcitonin, neurokinin A and
others. However,
previously it was demonstrated that PAM can also catalyze the formation of
alpha-amides from
glycinated substrates of non-peptide character, e.g. N-fatty acyl-glycines,
which are converted by PAM
to primary fatty acid amides (PFAMs) like oleamide. The identified and
purified peptidyl-glycine
amidating activities were shown to be dependent on copper and ascorbate
(Emeson et at. 1984. Journal
of Neuroscience: 2604-13; Kumar et al. 2016. J Mol Endocrinol 56(4):T63-76;
Wand et al. 1985.
Neuroendocrinology 41: 482-89).
In humans, the PAM gene is located at chromosome 5q21.1 having a length of 160
kb containing 25
known exons (Gaier et al. 2014. BMC Endocrine Disorders 14). At least 6
isoforms are known to be
generated by alternative splicing (SEQ ID 1-6). The PAM enzyme was found to be
expressed at different
levels in almost all mammalian cell types, with significant expression in
airway epithelium, endothelial
cells, ependymal cells in the brain, adult atrium, brain, kidney, pituitary,
gastrointestinal tract and
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2
reproductive tissues (Chen et al. 2018. Diabetes Obes Metab 20 S'uppl 2:64-76;
Oldham et al. 1992.
Biochem Biophys Res Commun 184(1): 323-29; Schafer etal. 1992. J Neurosci
12(1): 222-34).
However, the highest human PAM activity was described in the pituitary, the
stalk and hypothalamus.
The plasma amidating activity of healthy children below 15 years was
significantly higher than that of
healthy adults (Wand etal. 1985 Metabolism 34(11): 1044-52).
The precursor protein (1-973 amino acids) of the largest known PAM Isoform 1
(SEQ ID No. 1) encoded
by the PAM cDNA is depicted in Figure 1. The N-terminal signal sequence (amino
acids 1-20) assures
direction of the nascent PAM polypeptide into the secretory lumen of
endoplasmic reticulum and is
subsequently cleaved co-translationally. Afterwards the PAM-pro-peptide is
processed by the same
machinery used for the biosynthesis of integral membrane proteins and secreted
proteins including
cleavage of the pro-region (amino acids 21-30), assuring proper folding,
disulfide bond formation,
phosphatylation and glycosylation (Bousquet-Moore et al. 2010. J Neurosci Res
88(12):2535-45).
As depicted in Figure 1, the PAM cDNA further encodes two distinct enzymatic
activities. The first
enzymatic activity is named peptidyl-glycine alpha-hydroxylating monooxygenase
(PHM; EC
1.14.17.3), is an enzyme, capable of catalyzing the conversion of a C-terminal
glycine residue to an
alpha-hydroxy-glycine. The second activity is named peptidyl-a-hydroxy-glycine
alpha-amidating lyase
(PAL; EC 4.3.2.5) is an enzyme capable of catalyzing the conversion of an
alpha-hydroxy-glycine to an
alpha-amide with subsequent glyoxylate release. The sequential action of these
separate enzymatic
activities results in the overall peptidyl-glycine alpha amidating activity.
The first enzymatic activity
(PHM) is located directly upstream of the pro-region (within of amino acids 31-
494 of isoform 1 (SEQ
ID No. 7)). The second catalytic activity (PAL) is located after exon 16 in
isoform 1 within of amino
acids 495-817 (SEQ ID No. 8).
As depicted in Figure 2, both activities may be encoded together within of one
polypeptide as a
membrane-bound protein (isofonns 1, 2, 5, 6, corresponding to SEQ ID No. 1, 2,
5 and 6) as well within
of one polypeptide as a soluble protein lacking the transmembrane domain
(isoforms 3 and 4;
corresponding to SEQ ID No. 3 and 4). While isoforms 1, 2, 5 and 6 remain in
the outer plasma
membrane after fusion of secretory vesicles with the plasma membrane with
subsequent endocytosis
and recycling or degradation, soluble PAM isoforms lacking the TMD (isoforms 3
and 4) (amino acids
864-887) are co-secreted with the peptide-hormones (Wand etal. 1985 Metabolism
34(11): 1044-52).
Furthermore, prolionnone conyertases may convert membrane bound PAM protein
into soluble PAM
protein by cleavage within the flexible region (exons 25/26) connecting PAL
with the TMD during the
secretory pathway (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45).
The PHM subunit
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may be cleaved from soluble or membrane bound PAM within the secretory pathway
by prohormone
convertases that address a double-basic cleavage-site in the exon 16 region.
Furthermore, during
endocytosis the full-length PAM protein may be also converted into a soluble
fonii due to the action of
alpha- and gamma secretases (Bousquet-Moore et al. 2010. JNeurosci Res
88(12):2535-45). Membrane
bound PAM from late endosome can be further secreted in form of exosomal
vesicles.
PHM and PAL activities, as well as the activity of the full-length PAM were
determined in several
human tissues and body fluids. However, the separated PHM and PAL activities
in soluble forms will
also lead to formation of c-terminally alpha amidated products from c-
terminally glycinated substrates
when allowed to perform their separate reactions in the same compartment, body-
fluid or in vitro
experimental setup. How the transfer of the PHM hydroxylated product to the
PAL takes place is not
exactly understood to date. There is evidence that the hydroxylated product is
released into solution and
is not directly transferred from PHM to PAL (Yin et at. 2011. PLoS One
6(12):e28679). Also not clear
to date is the source of PAM in circulation.
The partial reaction of PHM is depicted in Figure 2. PHM is a copper dependent
monooxygenase
responsible for stereo-specific hydroxylation of the c-terminal glycine at the
alpha-carbon atom. During
the hydroxylation reaction ascorbate is believed to be the naturally occurring
reducing agent, while the
oxygen in the newly formed hydroxyl group was shown to originate from
molecular oxygen. The partial
reaction of the PAL is depicted in Figure 2. The catalytic action of PAL
involves proton abstraction
form the PHM-formed hydroxy-glycine by a protein-backbone derived base and a
nucleophilic attack
of hydroxyl-group oxygen to the divalent metal leading to a cleavage of
glyoxylate and formation of a
c-terminal amide.
Thus the term "amidating activity", "alpha-amidating activity", "peptidyl-
glycine alpha-amidating
activity" or "PAM activity" refers to the sequential enzymatic activities of
PHM and PAL, independent
of the present splice variant or mixtures of splice variants or post-
translationally modified PAM enzymes
or soluble, separated PHM or PAL activities or soluble PHM and membrane bound
PAL or
combinations of all mentioned forms leading to the formation of alpha amidated
products of peptide or
non-peptide character from glycinated substrates of peptide or non-peptide
character. In other words,
the term "amidating activity", "alpha-amidating activity", "peptidyl-glycine
alpha-amidating activity"
or -PAM activity" may be described as the sequential action of enzymatic
activities located within
amino acids 31 to 817 in the propeptide encoded by the human PAM cDNA,
independent of present
splice-variants or mixtures thereof.
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PAM activity was analyzed in several human tissues and body fluids of healthy
specimen or those
suffering from several diseases. To summarize efforts that has been done in
past:
Detection of PAM activities in human body-fluids mainly involves usage of
radiolabeled synthetic
tripeptides such as 125I-D-TyrValGly, '25I-N-acetyl-TyrValGly or comparably
modified tripeptides and
quantification of the amidated product due to gamma-scintillation (Kapuscinski
et al. 1993. Clinical
Endocrinology 39(1): 51-58; Wand etal. 1985 Metabolism 34(11): 1044-52;
Tsukamoto etal. 1995.
Internal Medicine 34(4): 229-32. Wand et al. 1987 Neurology 37: 1057-61. Wand
et al. 1985
Neuroendocrinol 41: 482-89). Furthermore, Substance P-Gly or a truncated
version Neuropeptide Y-
Gly were utilized as substrates for PAM activity assays (Gether etal. 1991 Mol
Cell Endocrinol 79 (1-
3): 53-63; Hyyppei et al. 1990 Pain 43: 163-68; Jeng et al. 1990 Analytical
Biochemistry 185(2): 213-
19).
The presence of alpha-amidating activity in human circulation was initially
proved by Wand et al. (Wand
etal. 1985 Metabolism 34(11): 1044-52). They reported no sex differences but
some variations of PAM
activity in certain disease states: Plasma PAM activities were increased in
hypothyroid adults as well as
in patients with medullary thyroid carcinoma. The activity of PAM in tissues
of medullary thyroid
carcinoma, pheochromocytoma and pancreatic islet tumors were shown to be
elevated suggesting
increased fonnation of amidated peptides in endocrine tumor tissues (Gether et
al. 1991 Mol Cell
Endocrinol 79 (1-3): 53-63; Wand etal. 1985 Neuroendocrinol 41: 482-89).
Patients suffering from multiple endocrine neoplasia type 1 (MEN-1) and
pernicious anemia showed a
decreased plasma PAM activity in comparison to healthy control subjects
(Kapuscinski et al. 1993. Clin
Endocrinol 39(1): 51-58).
The presence of amidating activity in human cerebrospinal fluid (CSF) was
shown by Wand and
colleagues (Wand et at. 1985 Neuroendocrinol 41: 482-89). In patients
suffering from Alzheimer's
disease (AD) plasma PAM activities were shown to be unaltered when compared to
healthy controls,
while CSF PAM activities were significantly decreased in comparison to
activities from normal
specimen _(Wand etal. 1987 Neurology 37: 1057-61). In addition, in
W02015/103594 the presence of
PAM-Protein in CSF detected by mass spectrometry of AD-patients was proposed
to be reduced
compared to healthy controls. Moreover, ADM-NH2, one of the amidated products
of PAM, was shown
to be reduced in patients with prevalent and incident Alzheimer's disease
(W02019/154900). However,
no direct association of circulating PAM activities were reported to date
being associated with
prediction, diagnosis or progression of AD.
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Amidating activity in CSF of patients with low back pain was analyzed using 1-
12 Substance P-Gly
(SP-Gly) as substrate (Hyyppa etal. 1990 Pain 43: 163-68). PAM activities of
patients suffering from
multiple sclerosis (MS) were shown to be increased in CSF, with a significant
decrease in serum
(Tsukamoto et at. 1995. Internal Medicine 34(4): 229-32; W02010/005387). An
association between
plasma activity of PAM and type-2-diabetes was described in (W02014/118634).
Even though some findings were made regarding PAM activity in human body
fluids and diseases or
disease progression, there is no information on PAM concentrations in human
body fluids, particularly
in the circulation, measured with an immunoassay. As shown in the Examples,
detection methods for
the determination of the level of PAM (as the total amount or the activity of
PAM) in a bodily fluid of
a subject were established. With these assays it was shown that the level of
PAM is decreased in a
number of diseases as well as in patients who will develop a disease.
Moreover, it was the surprising
finding of the present application that the in vivo administration of
recombinant PAM enzyme can be
used to increase the PAM-level in the circulation, which results in an
enhanced conversion of the PAM
substrate adrenomedullin-glycine to mature adrenomedullin-amid. In conclusion,
PAM may be used as
therapy in a subject.
Detailed Description of the Invention
Subject-matter of the present application is peptidylglycine alpha-amidating
monooxygenase (PAM) for
use as a medicament.
Further subject-matter of the present application is PAM for use as a
medicament for treatment of a
subject, wherein said treatment comprises:
reducing the potential or risk for a disease or disorder, and/ or
(ii) reducing the occurrence of a disease or disorder, and/ or
(iii) reducing the severity of a disease or disorder.
One embodiment of the present application relates to PAM for use as a
medicament for treatment of a
subject, wherein said disease or disorder is selected from the group
comprising dementia, cardiovascular
disorders, kidney diseases, cancer, inflammatory or infectious diseases and/or
metabolic diseases.
Another embodiment of the present application relates to PAM for use as a
medicament for treatment
of a subject, wherein said subject is characterized by
= a level of PAM and/or its isoforms and/or fragments thereof below a
threshold and/ or
= a peptide-Gly/ peptide-amide ratio above a threshold
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in a sample of bodily fluid of said subject.
Another specific embodiment of the present application relates to PAM for use
as a medicament for
treatment of a subject, wherein said peptide is selected from the group of
comprising adrenomedullin
(ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal
peptide (PAMP),
amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S.
neuromdin U,
calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid
polypeptide, chromogranin A,
insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin,
big gastrin, gastrin,
glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating
polypeptide (PACAP), secretin,
somatolibcrin, peptide histidinc mothioninc (PHM), vasoactivc intestinal
peptide (VIP), gonadoliberin,
kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P,
neurokinin A,
neurokinin B, peptide YY, pancreatic hormone, deltorphin 1, orexin A and B,
melanotropin alpha (alpha-
MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin,
vasopressin.
Another embodiment of the present application relates to PAM for use as a
medicament for treatment
of a subject, wherein said subject is characterized by
= an ADM-Gly/ bio-ADM ratio above a threshold and/ or
= a bio-ADM concentration below a threshold
in a bodily fluid of said patient.
Another preferred embodiment of the present application relates to PAM for use
as a medicament for
treatment of a subject, wherein the level of PAM and/or its isoforms and/or
fragments thereof is the total
concentration of PAM and/or its isoforms and/or fragments thereof having at
least 12 amino acids or the
activity of PAM and/or its isoforms and/or fragments thereof.
One embodiment of the present application relates to PAM for use as a
medicament for treatment of a
subject, wherein the total concentration of PAM and/or its isoforms and/or
fragments thereof having at
least 12 amino acids or the activity of PAM and/or its isoforms and/or
fragments thereof is selected from
the group comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4,
SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.
Another embodiment of the present application relates to PAM for use as a
medicament for treatment
of a subject, wherein the sample of bodily fluid of said subject is selected
from the group of blood,
serum, plasma, urine, cerebrospinal fluid (CSF), and saliva.
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Another specific embodiment of the present application relates to PAM for use
as a medicament,
wherein said PAM is selected from the group comprising isolated and/ or
recombinant and/or chimeric
PAM.
One embodiment of the present application relates to PAM for use as a
medicament, wherein said
recombinant PAM is selected from the sequences comprising SEQ ID No. 1, SEQ ID
No. 2, SEQ ID
No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No, 7, SEQ ID No. 8
and SEQ ID No. 10.
One embodiment of the present application relates to PAM for use as a
medicament in a subject having
a level of PAM and/or its isoforms and/or fragments thereof below a threshold
and/ or a peptide-Gly/
peptide-amide ratio above a threshold in a sample of bodily fluid of said
subject.
One preferred embodiment of the present application relates to PAM for use as
a medicament in a subject
identified as having a level of PAM and/or its isoforms and/or fragments
thereof below a threshold and/
or a peptide-Gly/ peptide-amide ratio above a threshold in a sample of bodily
fluid of said subject.
One embodiment of the present application relates to PAM for use as a
medicament, wherein said use
comprises testing a subject whether the subject has a level of PAM and/or its
isoforms and/or
fragments thereof below a threshold and/ or a peptide-Gly/ peptide-amide ratio
above a threshold in a
sample of bodily fluid of said subject., and providing treatment with PAM if
the subject is identified
as having risk for a disease or disorder.
One embodiment of the present application relates to PAM for use as a
medicament, wherein PAM is
combined with ascorbate and/ or copper.
Subject-matter of the present application is also a pharmaceutical formulation
comprising
peptidylglycine alpha-amidating monooxygenase (PAM).
One embodiment of the present application relates to a pharmaceutical
formulation comprising PAM,
wherein said pharmaceutical formulation is administered orally (e.g.
inhalation), epicutaneously,
subcutaneously, intradermally, sublingually, intramuscularly, intraarterially,
intravenously, or via the
central nervous system (CNS, intracerebrally, intracerebroventricularly,
intrathecally) or via
intrapefitoneal admini strati on.
One preferred embodiment of the present application relates to a
pharmaceutical formulation, wherein
said pharmaceutical formulation is a solution, preferably a ready-to-use
solution.
Another embodiment of the present application relates to a pharmaceutical
formulation, wherein said
pharmaceutical formulation is in a freeze-dried state.
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Another embodiment of the present application relates to a pharmaceutical
formulation, wherein said
pharmaceutical formulation is administered intra-muscular.
Another specific embodiment of the present application relates to a
pharmaceutical formulation, wherein
said pharmaceutical formulation is administered intra-vascular.
Another preferred embodiment of the present application relates to a
pharmaceutical formulation,
wherein said pharmaceutical formulation is administered via infusion.
One embodiment of the present application relates to a pharmaceutical
formulation, wherein said
pharmaceutical formulation is to be administered systemically.
Another embodiment of the present application relates to a pharmaceutical
formulation, the formulation
comprising PAM and/or optionally one or more pharmaceutically acceptable
ingredients
Another preferred embodiment of the present application relates to a
pharmaceutical formulation, the
formulation comprising PAM, ascorbate and/ or copper.
Another embodiment of the present application relates to a pharmaceutical
formulation, the formulation
comprising PAM in combination with ascorbate and/ or copper.
Subject-matter of the present application is also a method of treatment in a
subject, the method
comprising administering PAM to said subject, the method further comprising
i. reducing the potential or risk for a disease or disorder,
and/ or
reducing the occurrence of a disease or disorder, and/ or
reducing the severity of a disease or disorder.
One embodiment of the present application relates to a method of treatment in
a subject, wherein said
disease or disorder is selected from the group comprising dementia,
cardiovascular disorders, kidney
diseases, cancer, inflammatory or infectious diseases and/or metabolic
diseases.
Another embodiment of the present application relates to a method of treatment
in a subject, wherein
said subject is characterized by
= a level of PAM and/or its isoforms and/or fragments thereof below a
threshold and/ or
= a peptide-Gly/ peptide-amide ratio above a threshold
in a sample of bodily fluid of said subject.
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Another preferred embodiment of the present application relates to a method of
treatment in a subject,
wherein the level of PAM and/or its isoforms and/or fragments thereof is the
total concentration of PAM
and/or its isoforms and/or fragments thereof having at least 12 amino acids or
the activity of PAM and/or
its isoforms and/or fragments thereof
One embodiment of the present application relates to a method of treatment in
a subject, wherein the
total concentration of PAM and/or its isoforms and/or fragments thereof having
at least 12 amino acids
or the activity of PAM and/or its isoforms and/or fragments thereof is
selected from the group
comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6,
SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.
Another embodiment of the present application relates to a mcthod of treatment
in a subject, wherein
the sample of bodily fluid of said subject is selected from the group of
blood, serum, plasma, urine,
cerebrospinal fluid (CSF), and saliva.
Another preferred embodiment of the present application relates to a method of
treatment in a subject,
wherein said PAM is selected from the group comprising isolated and/ or
recombinant and/or chimeric
PAM.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein the level of PAM
and/or its isoforms and/or fragments thereof is the total concentration of PAM
and/or its isoforms and/or
fragments thereof having at least 12 amino acids or the activity of PAM and/or
its isoforms and/or
fragments thereof in a sample of bodily fluid of said subject.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein the activity of PAM
and/or its isofonns and/or fragments thereof in a sample of bodily fluid in
said subject is selected from
the group comprising the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,
SEQ ID No. 4, SEQ
ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein the total
concentration of PAM and/or its isofonns and/or fragments thereof having at
least 12 amino acids in a
sample of bodily fluid in said subject is detected with an immunoassay.
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One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein the activity of PAM
and/or its isofonns and/or fragments thereof in a sample of bodily fluid in
said subject is detected using
a peptide-Gly as substrate.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein the activity of PAM
and/or its isoforms and/or fragments thereof in a sample of bodily fluid in
said subject is detected using
a peptide-Gly as substrate and wherein the peptide-Gly substrate is selected
from the group comprising
adrcnomcdullin (ADM), adrcnomedullin-2, intermcdin-short, pro-adrcnomedullin N-
20 terminal
peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B,
neuromedin S,
ncuromdin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet
amyloid polypeptide,
chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP),
cholecystokinin, big gastrin,
gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-
activating polypeptide (PACAP),
secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive
intestinal peptide (VIP),
gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide
gamma, substance P,
neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I,
orexin A and B,
melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing
hormone (TRH),
oxytocin and vasopressin.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for useas a medicament for treatment of a subject, wherein
the level of PAM
and/ or isoforms and/ or fragments thereof in a bodily fluid sample of said
subject is measured using an
assay, wherein said assay is comprising two binders that bind to two different
regions of PAM, wherein
the two binders are directed to an epitope of at least 5 amino acids,
preferably at least 4 amino acids in
length, wherein said two binders are directed to an epitope comprised within
the following sequences
of PAM: peptide 1 (SEQ ID No. 11), peptide 2 (SEQ ID No. 12), peptide (SEQ ID
No. 13), peptide 4
(SEQ ID No. 14), peptide 5 (SEQ ID No. 15), peptide 6 (SEQ ID No. 16), peptide
7 (SEQ ID No. 17),
peptide 8 (SEQ ID No. 18), peptide 9 (SEQ ID No. 19), peptide 10 (SEQ ID No.
20), peptide 11 (SEQ
ID No. 21), peptide 12 (SEQ ID No. 22), peptide 13 (SEQ ID No. 23) peptide 14
(SEQ ID No. 24) and
recombinant PAM (SEQ ID No. 10).
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein the method for
determining the activity of PAM and/ or isoforms or fragments thereof in a
bodily fluid sample of a
subject is comprising the steps:
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= contacting said sample with a capture-binder that binds specifically to
active full-length
PAM, its isoforms and/ or active fragments thereof,
= separating PAM bound to said capture-binder
= adding a substrate of PAM to said separated PAM
quantifying PAM activity by measuring the conversion of the substrate of PAM
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for uscas a medicament for treatment of a subject,
wherein the method for determining the activity of PAM and/ or isoforms and/
or fragments thereof in
a bodily fluid sample of a subject is comprising the steps:
= contacting said sample with a substrate (peptide-Gly) of PAM for an
interval of time at t=0 min
and t=n+1 min
= detecting the reaction product (alpha-amidated peptide) of PAM in said
sample at t=0 min and
t=n+1 min, and
= quantifying the activity of PAM by calculating the difference of the
reaction product between
t=0 and t=n+1.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for useas a medicament for treatment of a subject,
wherein said binders are selected from the group comprising an antibody, an
antibody fragment or a
non-Ig scaffold.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein said subject is characterized by
= a level of PAM and/or its isoforms and/or fragments thereof below a
threshold and/ or
= a peptide-Gly/ peptide-amide ratio above a threshold
in a bodily fluid of said subject.
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a medicament for treatment of a subject,
wherein said subject is characterized by
= an ADM-Gly/ bio-ADM ratio above a threshold and/ or
= a bio-ADM concentration below a threshold
in a bodily fluid of said patient.
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The threshold is pre-determined by measuring the level of PAM and/or its
isoforms and/or fragments
and/ or ADM-NH2 thereof in healthy controls and calculating e.g., the
according 25-percentile, more
preferably the 10-percentile, even more preferably the 5-percentile. The lower
boarder of the 25-
percentile, more preferably the 10-percentile, even more preferably the 5-
percentile, defines the
threshold for healthy versus diseased patients or healthy versus subjects at
risk of getting a disease or
subjects not at risk of getting an adverse event versus subjects at risk of
getting an adverse event, if the
level of said diseased subjects or subjects at risk of getting a disease or
adverse event is below a
threshold. The level of PAM and/or its isoforms and/or fragments thereof may
be detected as total PAM
concentration and/ or PAM activity. In relation to said percentiles, the lower
threshold that divides
between healthy and diseased patients or healthy versus subjects at risk of
getting a disease or subjects
not at risk of getting an adverse event versus subjects at risk of getting an
adverse event by detecting the
PAM activity in plasma may be between 15 and 8 ug/(L*11) or below, more
preferably between 13.5
and 8 ug/(L*h) or below, even more preferred between 10.5 and 8 ug/(L*h) or
below, most preferred
below 8 ug/(L*h), PAM activity in serum may be between 10 and 5 ug/(L*h) or
below, more preferably
between 8 and 5 ug/(L*h) or below, most preferred below 5 ug/(L*h) using a PAM
activity assay.
In relation to said percentiles, the lower threshold that divides between
healthy and diseased patients or
healthy versus subjects at risk of getting a disease or subjects not at risk
of getting an adverse event
versus subjects at risk of getting an adverse event by detecting ADM-NH2 is
equal or below 15 pg/ml,
preferably equal or below 10 pg/ml, preferably equal or below 5 pg/mL.
The threshold is pre-determined by measuring the ratio of ADM-Glv to ADM-NH2
in healthy controls
and calculating e.g., the according 75-percentile, more preferably the 90-
percentile, even more
preferably the 95-percentile. The upper boarder of the 75-percentile, more
preferably the 90-percentile,
even more preferably the 95-percentile, defines the threshold for healthy
versus diseased patients or
healthy versus subjects at risk of getting a disease or subjects not at risk
of getting an adverse event
versus subjects at risk of getting an adverse event, if the level of said
diseased subjects or subjects at
risk of getting a disease or adverse event is above a threshold. In relation
to said percentiles, the upper
threshold that divides between healthy and diseased patients or healthy versus
subjects at risk of getting
a disease or subjects not at risk of getting an adverse event versus subjects
at risk of getting an adverse
event by detecting the ration of ADM-Gly to ADM-NH?, wherein the ADM-Gly/ ADM-
NH2 ratio is in
a range between 1 and 10, preferably between 1.5 and 7.5, preferably between 2
and 5, most preferred
the threshold is 2.5.
The predetermined value can vary among particular populations selected,
depending on certain factors,
such as gender, age, genetics, habits, ethnicity or alike.
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The person skilled in the art knows how to determine thresholds from conducted
previous studies. The
person skilled in the art knows that a specific threshold value may depend on
the cohort used for
calculating a pre-determined threshold that can be later-on used in routine.
The person skilled in the art
knows that a specific threshold value may depend on the calibration used in
the assay. The person skilled
in the art knows that a specific threshold value may depend on the sensitivity
and/or specificity that
seems to be acceptable for the practitioner.
The sensitivity and specificity of a diagnostic test depends on more than just
the analytical "quality" of
the test, they also depend on the definition of what constitutes an abnormal
result. In practice, Receiver
Operating Characteristic curves (ROC curves), arc typically calculated by
plotting the value of a variable
versus its relative frequency in "normal" (i.e., apparently healthy) and
"disease" populations (i.e.,
patients suffering from an infection). Depending on the particular diagnostic
question to be addressed,
the reference group must not be necessarily "normal", but it might be a group
of patients suffering from
another disease, from which the diseased group of interest shall be
differentiated. For any particular
marker, a distribution of marker levels for subjects with and without a
disease will likely overlap. Under
such conditions, a test does not absolutely distinguish normal from disease
with 100% accuracy, and the
area of overlap indicates where the test cannot distinguish normal from
disease. A threshold is selected,
above which (or below which, depending on how a marker changes with the
disease) the test is
considered to be abnormal and below which the test is considered to be normal.
The area under the ROC
curve is a measure of the probability that the perceived measurement will
allow correct identification of
a disease. ROC curves can be used even when test results do not necessarily
give an accurate number.
As long as one can rank results, one can create a ROC curve. For example,
results of a test on "disease"
samples might be ranked according to degree (e.g,. 1=low, 2=normal, and
3=high). This ranking can be
correlated to results in the "normal" population, and a ROC curve created.
These methods are well
known in the art (see, e.g.. Hartley et al, 1982). Preferably, a threshold is
selected to provide a ROC
curve area of greater than about 0.5, more preferably greater than about 0.7.
The term "about" in this
context refers to +/- 5% of a given measurement.
Once the threshold value is determined by using a previous study cohort and
taking into consideration
all the above-mentioned points the medical practitioner will use the pre-
determined threshold for
diagnosing or prognosing a disease and/ or predicting a risk of getting a
disease or an adverse event in
a subject and will determine whether the subject has a value above or below
said pre-determined
threshold value in order to make an appropriate diagnosis, prognosis,
prediction or monitoring.
The mentioned threshold values above might be different in other assays, if
these have been calibrated
differently from the assay system used in the present invention. Therefore,
the mentioned threshold(s)
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shall apply for such differently calibrated assays accordingly, taking into
account the differences in
calibration. One possibility of quantifying the difference in calibration is a
method comparison analysis
(correlation) of the assay in question (e.g. PAM assay) with the respective
biomarker assay used in the
present invention by measuring the respective biomarker or it's activity (e.g.
PAM) in samples using
both methods. Another possibility is to determine with the assay in question,
given this test has sufficient
analytical sensitivity, the median biomarker level of a representative non-nal
population, compare results
with the median biomarker levels with another assay and recalculate the
calibration based on the
difference obtained by this comparison. With the calibration used in the
present invention, samples from
normal (healthy) subjects have been measured: the median plasma PAM activity
was 18.4 pg/(L*h)
(inter quartile range lIQR1 13.5 ¨ 21.9 pg/(L*h)), the median scrum PAM
activity was 11.0 ng/(L*h)
(inter quartile range ['OR] 8.1 ¨ 13.1 pg/(L*h). In samples from normal
(healthy) subjects have ADM-
NH2 been measured: median plasma bio-ADM (mature ADM-NH2) was 13.7 pg/ml
(inter quartile range
['OR] 9.6¨ 18.7 pg/mL) (Weber et al. 2017. JALIVI, 2(2): 222-233).
One preferred embodiment of the present application relates to peptidylglycine
alpha-amidating
monooxygenase (PAM) for use as a therapy in a subject,
wherein said subject is a healthy subject that has been predicted as having an
increased risk to develop
a disease or disorder.
Another preferred embodiment of the present application relates to
peptidylglycine alpha-amidating
monooxygenase (PAM) for use as a therapy in a subject,
wherein said subject is a healthy subject that has been predicted as having an
increased risk to develop
a disease or disorder or an adverse event in the future.
The term "PAM" of the present disclosure refers to isolated PAM, including
splice variants, isoforms,
and polymorphic forms thereof. Also included are recombinant PAM (RecPAM) and
chimeric PAM. In
specific aspects, the PAM is RecPAM. The amino acid sequence of PAM isoform 1
to 6 is shown in
SEQ ID No. 1 to 6. In some aspects, PAM disclosed herein has at least about
70%, at least about 75%,
at least about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 96%, at
least about 97%, at least about 98%, or at least about 99% sequence identity
to the amino acid sequence
of SEQ ID No.: 1 to 6.
It is to be understood by the skilled artisan, that the PAM isoform sequences
(SEQ ID No. 1 to 6) as
represented in the sequence list, contain an N-terminal signal sequence (amino
acid 1-20). This N-
terminal signal sequence is cleaved off prior to secretion of the protein.
Therefore, in a preferred
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embodiment the PAM isoform sequences (SEQ ID No. 1 to 6) and/ or fragments
thereof do not contain
the N-terminal signal sequence.
In some aspects, the PAM is a functional fragment (i.e., PHM (SEQ ID No. 7)
and PAL (SEQ ID No.
8), PAM conserving at least about 10%, at least about 20%, at least about 30%,
at least about 40%, at
least about 50%, at least about 60%, at least 70%, at least about 80%, or at
least about 90% of the PAM
activity of the corresponding functional fragment of PAM. In some aspects, the
PAM is a variant or a
derivative of PAM disclosed herein.
The percentage of identity of an amino acid or nucleic acid sequence, or the
term "% sequence identity",
is defined herein as the percentage of residues in a candidate amino acid or
nucleic acid sequence that
is identical with the residues in a reference sequence after aligning the two
sequences and introducing
gaps, if necessary, to achieve the maximum percent identity. In a preferred
embodiment, the calculation
of said at least percentage of sequence identity is carried out without
introducing gaps. Methods and
computer programs for the alignment are well known in the art, for example -
Align 2" or the BLAST
service of the National Center for Biotechnology Information (NCBI).
PAM for use according to the present disclosure can be a commercial PAM
enzyme, or any composition
comprising the PAM enzyme and any means capable of producing a functional PAM
enzyme in the
context of the current invention, such as DNA or RNA nucleic acids encoding a
PAM protein. The
nucleic acid encoding PAM may be embedded in suitable vectors such as
plasmids, phagemids, phages,
(retro)viruses, transposons, gene therapy vectors and other vectors capable of
inducing or conferring
production of PAM. Also native or recombinant microorganisms, such as
bacteria, fungi, protozoa and
yeast may be applied as a source of PAM in the context of the current
disclosure.
In some aspects, the mammalian PAM is a human or a bovine PAM.
As used herein the terms "treat", "treatment", "treatment of', "therapy" or
"therapy of" refers to (i)
reducing the potential or risk for a disease or disorder, e.g. dementia,
cardiovascular disorders, kidney
diseases, cancer, inflammatory or infectious diseases and/or metabolic
diseases, (ii) reducing the
occurrence of a disease or disorder, e.g. dementia, cardiovascular disorders,
kidney diseases, cancer,
inflammatory or infectious diseases and/or metabolic diseases, (iii) reducing
the severity (e.g.,
ameliorating the symptoms) of a disease or disorder, e.g. dementia,
cardiovascular disorders, kidney
diseases, cancer, inflammatory or infectious diseases and/or metabolic
diseases, or (iv) a combination
thereof.
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The terms "subject" or "patient" as used herein refer to any subject,
particularly a mammalian subject,
for whom therapy or prognosis of a disease is desired. As used herein, the
terms "subject or "patient"
include any human or nonhuman animal. As used herein, phrases such as "a
patient having a disease"
includes subjects, such as mammalian subjects, that would benefit from the
administration of a therapy
with PAM, as disclosed herein.
In some aspects of the present disclosure, a therapeutic agent for the
treatment or prevention a disease,
e.g., dementia, cardiovascular disorders, kidney diseases, cancer,
inflammatory or infectious diseases
and/or metabolic diseases, can comprise PAM, e.g., RecPAM or isolated PAM;
alone or in combination
with one or more standard therapeutic agents generally used for the treatment
or prevention of a disease,
e.g., dementia, cardiovascular disorders, kidney diseases, cancer,
inflammatory or infectious diseases
and/or metabolic diseases.
"Therapeutically effective amount" means level or amount of therapeutic agent
that is aimed at, without
causing significant negative or adverse side effects to the target, (1)
delaying or preventing the onset of
the target disease, disorder, or condition; (2) slowing down or stopping the
progression, aggravation, or
deterioration of one or more symptoms of the target disease, disorder, or
condition; (3) bringing about
ameliorations of the symptoms of the target disease, disorder, or condition;
(4) reducing the severity or
incidence of the target disease, disorder, or condition; or (5) curing the
target disease, disorder, or
condition. A therapeutically effective amount may be administered prior to the
onset of the target
disease, disorder, or condition, for a prophylactic or preventive action.
Alternatively, or additionally,
the therapeutically effective amount may be administered after initiation of
the target disease, disorder,
or condition, for a therapeutic action.
In some aspects, the PAM (e.g., RecPAM) is administered as doses of at least
about 500 U/kg, at least
about 600 U/kg, at least about 700 U/kg, at least about 800 U/kg, at least
about 900 U/kg, at least about
1000 U/kg, at least about 1100 U/kg, at least about 1200 U/kg, at least about
1300 U/kg, at least about
1400 U/kg, at least about 1500 U/kg, at least about 1600 U/kg. at least about
1700 U/kg, at least about
1800 U/kg, at least about 1900 U/kg. or at least about 2000 U/kg per dose. In
some aspects, the PAM
(e.g. RecPAM) is administered as doses above 2000 U/kg per dose. In some
aspects, the PAM (e.g.,
RecPAM) is administered as doses below 500 U/kg per dose.
In some aspects, the PAM (e.g., RecPAM) is administered at a dose between
about 500 U/kg and about
1500 U/kg, between about 600 U/kg and about 1400 U/kg, between about 700 U/kg
and about 1300
U/kg, between about 800 U/kg and about 1200 U/kg, or between about 900 U/kg
and about 1100 U/kg.
In some specific aspects, PAM is administered as about 1000 U/kg doses.
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In some aspects, the PAM is a human or bovine PAM. In some aspects, the PAM is
a recombinant PAM
(RecPAM). In some aspects, the PAM is a chimeric PAM. In a particular aspect,
the chimeric PAM is
RecPAM (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8). In some aspects, PAM disclosed
herein has at least about
70%, at least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%,
at least about 96%, at least about 97%, at least about 98%, or at least about
99% sequence identity to
the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, S.
In some aspects, the PAM is a functional fragment (i.e., PHM (SEQ ID No. 7)
and PAL (SEQ ID No.
8), (i.e., a fragment of the PAM, e.g., PAM conserving at least about 10%, at
least about 20%, at least
about 30%, at least 40%, at least about 50%, at least about 60%, at least 70%,
at least about 80%, or at
least about 90% of the PAM activity of the corresponding full- length PAM). In
some aspccts, the PAM
is a variant or a derivative of a PAM disclosed herein.
PAM is administered at a dose that significantly increases the concentration
of bio-ADM in the blood
of a patient. A significant increase in the concentration of bio-ADM is
defined as an increase of about
10%, more preferred of about 25 %, even more preferred of about 50%, even more
preferred of about
100%, even more preferred of about 200%, even more preferred of about 300%,
even more preferred of
about 500%, most preferred up to 1000%. It is preferred that the concentration
of bio-ADM is increased
to the median concentration of a healthy population. Samples from normal
(healthy) subjects have been
measured: median plasma bio-ADM (mature ADM-NH2) was 24.7 pg/ml, the lowest
value 11 pg/ml
and the 99th percentile 43 pg/ml Marino et al. 2014. Critical Care 18:R34)."
In some aspects, the PAM is RecPAM and it is administered at a dose of at
least about 0.1 mg/kg, at
least about 0.2 mg/kg, at least about 0.3 mg/kg, at least about 0.4 mg/kg, at
least about 0.5 mg/kg, at
least about 0.6 mg/kg, at least about 0.7 mg/kg, at least about 0.8 mg/kg, at
least about 0.9 mg/kg, at
least about 1 mg/kg, at least about 1.1 mg/kg, at least about 1.3 mg/kg, at
least about 1.4 mg/kg, at least
about 1.5 mg/kg, at least about 1.6 ing/kg, at least about 1.7 ing/kg, at
least about 1.8 mg/kg, at least
about 1.9 mg/kg, at least about 2 mg/kg, at least about 2.1 mg/kg, at least
about 2.2 mg/kg, at least about
2.3 mg/kg, or at least about 2.4/kg per dose. In some aspects, the PAM is
administered as doses above
2.4 mg/kg per dose.
In some aspects, the PAM is RecPAM, and it is administered at a dose of at
least about 100 U/kg, at
least about 200 U/kg, at least about 300 U/kg. at least about 400 U/kg, at
least about 500 U/kg, at least
about 600 U/kg, at least about 700 U/kg, at least about 800 U/kg, at least
about 900 U/kg, at least about
1000 U/kg, at least about 1100 U/kg, at least about 1200 U/kg, at least about
1300 U/kg, at least about
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1400 U/kg, at least about 1500 U/kg, at least about 1600 U/kg, at least about
1700 U/kg, at least about
1800 U/kg, at least about 1900 U/kg, or at least about 2000 U/kg. The PAM is
RecPAM, and it is
administered at a dose below 100 U/kg, the PAM is RecPAM, and it is
administered at a dose above
2000 U/kg.
In some aspects, the PAM is RecPAM and it is administered at a dose between
about 0.8 mg/kg and
about 2.4 mg/kg, between about 0.9 mg/kg and about 2.3 mg/kg, between about 1
mg/kg and about 2.2
mg/kg, between about 1.1 mg/kg and about 2.1 mg/kg, between about 1.2 mg/kg
and about 2 mg/kg,
between about 1.3 mg/kg and about 1.9 mg/kg, between about 1.4 mg/kg and about
1.8 mg/kg, or
between about 1.5 mg/kg and about 1.7 mg/kg. In some specific aspects, PAM is
administered as about
1.6 mg/kg doses.
In some aspects, the PAM is RecPAM and it has a specific activity of at least
about 100 U/mg, at least
about 200 U/mg, at least about 300 U/mg, at least about 400 U/mg, at least
about 500 U /mg, at least
about 600 U/mg, at least about 700 U/mg, at least about 800 U/mg, at least
about 900 U/mg, at least
about 1000 U/mg, at least about 1100 U/mg, at least about 1200 U/mg, at least
about 1300 U/mg, at
least about 1400 U/mg, at least about 1500 U/mg, at least about 1600 U/mg, at
least about 1700 U/mg,
at least about 1800 U/mg, at least about 1900 U/mg, or at least about 2000
U/mg.
In some aspects, the PAM is RecPAM and it has a specific activity of about
1000 U/ mg. In some
aspects, the PAM is RecPAM and it has a specific activity between about 600
U/mg and about 700
U/mg, or between about 500 U/mg and about 800 U/mg, or between about 400 U/mg
and about 900
U/mg, or between about 300 U/mg and about 1000 U/mg, or between about 200 U/mg
and about 1100
U/mg, or between 100 U/mg and about 1200 U/mg. In some aspects, the PAM is
RecPAM and it has a
specific activity below 100 U/mg. In some aspects, the PAM is RecPAM and it
has a specific activity
above 1200 U/mg.
In some aspects, only one dose of PAM (e.g., RecPAM) is administered per
treatment (e.g., one dose
per day for 1-7 days). In other aspects, more than one dose of PAM is
administered. In some aspects,
two, three, four, five, six, seven, eight, nine, 10, 1 1, 12, 13, 14, 15, 16,
17, 18, 19, 20, or 21 doses of
PAM are administered (e.g., at least two doses per day for 1-7 days).
In some aspects, the PAM doses are administered daily. In other aspects, PAM
doses are administered
every 2, 3, 4, 5, 6 or 7 days.
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In some aspects, a single dose is administered every day. In some aspects, 2,
3, or more doses are
administered every day.
In some aspects, the treatment with PAM is less than about 7 days. In some
aspects, the treatment with
PAM is less than 6 days, less than 5 days, less than 4 days, less than 3 days,
less than 2 days, or less
than 1 day.
In some aspects the treatment of PAM is more than 7 days, more than 14 days,
more than 21 days, more
than 28 days, more than 1 month, more than 3 months, more than 6 months, more
than 12 months, more
than 2 years, more than 5 years or more than 10 years.
In some aspects, the second measurement is conducted 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, or at 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or 16 weeks, or at intervening times, after administering the PAM, e.g.,
RecPAM.
The formulation, dosage regimen, and route of administration of a PAM, e.g.,
RecPAM, can be adjusted
to provide an effective amount for an optimum therapeutic response according
to the method disclosed
herein. With regard to the administration of PAM, the PAM may be administered
through any suitable
means, compositions and routes known in the art. With regard to dosage
regiments, a single bolus can
be administered, several divided doses can be administered over time or the
dose can be proportionally
reduced or increased as indicated by the exigencies of the therapeutic
situation.
The present disclosure provides a method of determining whether to treat a
patient having a disease or
preventing a disease with a therapeutic regimen comprising the administration
of PAM, wherein the
method comprises:
(a) measuring
= the level of PAM and/or its isoforrns and/or fragments thereof and/ or
= the peptide-Gly/ peptide-amide ratio
in a bodily fluid of said patient, and
(b) treating the patient, or suspending the treatment with a therapeutic
regimen comprising the
administration of PAM, e.g., isolated or recombinant PAM, if the patient is
determined to have
higher or lower concentrations or activities in the sample compared to a
predetermined threshold
level or levels, or compared to the level or levels in one or more controls.
The peptide-Gly may be selected from the group comprising adrenomedullin
(ADM), adrenomedullin-
intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin,
gastrin-releasing
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peptide, neuromedin C, neuromedin B, neuromedin S, neuromdin U, calcitonin,
calcitonin gene-related
peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin,
pancreastatin, prolactin-
releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like
peptide 1 (GLP-1),
pituitary adenylate cyclase-activating polypeptide (PACAP), secretin,
somatoliberin, peptide histidine
methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin,
kisspeptin, MIF-1, metastin,
neuropeptide K, neuropeptide gamma, substance P. neurokinin A, neurokinin B,
peptide YY, pancreatic
hormone, deltorphin 1, orexin A and B, melanotropin alpha (alpha-MSH),
melanotropin gamma,
thyrotropin-releasing hormone (TRH), oxytocin, vasopressin.
In a preferred embodiment said peptide-Gly is ADM-Gly and said peptide-amide
is ADM-NH2.
As used herein, the term diseases or disorders include all "PAM-related"
diseases or disorders that are
known now, or that will be found in the future, to be associated with a
decrease in PAM activity and/or
increase in the peptide-Gly/ peptide-amide ratio.
In a specific embodiment of said disease or disorder is selected from the
group comprising:
= dementia, wherein said dementia is selected from the group comprising
mild cognitive impairment
(MCI), Alzheimer's disease, vascular dementia, mixed Alzheimer's disease and
vascular
dementia, Lewy body dementia, frontotemporal dementia, focal dementias
(including progressive
aphasia), subcortical dementias (including Parkinson's disease) and secondary
causes of dementia
syndrome (including intracranial lesions).
= cardiovascular disorders, wherein said cardiovascular disorders may be
selected from a group
comprising atherosclerosis, hypertension, heart failure (including acute and
acute decompensated
heart failure), atrial fibrillation, cardiovascular ischemia, cerebral
ischemic injury, cardiogenic
shock, stroke (including ischemic and hemorrhagic stroke and transient
ischemic attack) and
myocardial infarction,
= kidney diseases, wherein said kidney diseases may be selected from a
group comprising renal
toxicity (drug-induced kidney disease), acute kidney injury (AKI), chronic
kidney disease (CKD),
diabetic nephropathy, end-stage renal disease (ESRD),
= cancer, wherein said cancer may be selected from a group comprising
prostate cancer, breast
cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer,
cervical cancer, skin cancer
(including melanoma), stomach cancer, liver cancer, pancreatic cancer,
leukemia, non-hodgkin's
lymphoma, kidney cancer, esophagus cancer, pharyngeal cancer,
= infectious diseases caused by infectious organisms such as bacteria,
vinises, fungi or parasites,
said infectious disease is selected from the group comprising SIRS, sepsis,
and septic shock.
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= metabolic diseases selected from the group comprising diabetes type 1,
diabetes type 2, metabolic
syndrome.
In one embodiment of the present application said disease is dementia and said
dementia is selected
from the group comprising mild cognitive impairment (MCI), Alzheimer's
disease, vascular dementia,
mixed Alzheimer's disease and vascular dementia, Lewy body dementia,
frontotemporal dementia, focal
dementias (including progressive aphasia), subcortical dementias (including
Parkinson's disease) and
secondary causes of dementia syndrome (including intracranial lesions).
In a specific embodiment said dementia is Alzheimer's disease.
In one embodiment of the present application said disease is cancer and said
cancer is selected from the
group comprising prostate cancer, breast cancer, lung cancer, colorectal
cancer, bladder cancer, ovarian
cancer, cervical cancer, skin cancer (including melanoma), stomach cancer,
liver cancer, leukemia, non-
hodgkin's lymphoma, kidney cancer, esophagus cancer and pharyngeal cancer.
In a specific embodiment said cancer is colorectal cancer.
In one embodiment of the present application said disease is a cardiovascular
disorder, wherein said
cardiovascular disorder is selected from a group comprising atherosclerosis,
hypertension, heart failure
(including acute and acute decompensated heart failure), atrial fibrillation,
cardiovascular ischemia,
cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and
hemorrhagic stroke and
transient ischemic attack) and myocardial infarction.
In a specific embodiment said cardiovascular disorder is heart failure
(including acute and acute
decompensated heart failure).
In another specific embodiment said cardiovascular disorder is stroke stroke
(including ischemic and
haemorrhagic stroke and transient ischemic attack) and myocardial infarction.
In another specific embodiment said cardiovascular disorder is atrial
fibrillation.
In another specific embodiment of the present application said disease is
SIRS, sepsis or septic shock.
In another specific embodiment of the present application said disease is
diabetes type 1, diabetes type
2, metabolic syndrome.
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In one embodiment, the patients with -PAM-related" disease or disorder is a
patient with chronically
reduced bio-ADM concentrations which may suffer from a subclinical endothelial
dysfunction.
Subclinical endothelial dysfunction e.g., in the brain may lead to dementia
especially Alzheimer's
disease.
The bodily fluid in the context of the present invention maybe selected from
the group of blood, serum,
plasma, cerebrospinal fluid (CSF), urine, saliva, sputum, and pleural
effusions. In a specific embodiment
of said method said sample is selected from the group comprising whole blood,
serum and plasma.
The term "pharmaceutical formulation" as used herein refers to a preparation
which is in such form as
to permit the biological activity of an active ingredient contained therein to
be effective, and which
contains no additional components which arc unacceptably toxic to a subject to
which the formulation
would be administered.
The present invention also relates to a pharmaceutical formulation comprising
a therapeutically effective
dose of PAM, e.g., RecPAM, in combination with at least one pharmaceutically
acceptable excipient.
"Pharmaceutically acceptable excipient" refers to an excipient that does not
produce an adverse, allergic
or other untoward reaction when administered to a subject. It includes in
addition to a therapeutic
protein, carriers, various diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other materials well
known in the art. The characteristics of the carrier will depend on the route
of administration.
In one embodiment of the present invention said pharmaceutical formulation is
administered orally (e.g.,
inhalation), epicutaneously, subcutaneously, intradermally, sublingually,
intramuscularly,
intraarterially, intravenously, or via the central nervous system (CNS,
intracerebrally,
intracerebroventricularly, intrathecally) or via intraperitoneal
administration.
For administration by inhalation, the compound is delivered in the form of an
aerosol spray from a
pressurized container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide
or a nebulizer.
Subject matter of the present invention is a pharmaceutical formulation of PAM
for use in therapy of a
subject according to the present invention, wherein said pharmaceutical
formulation is a solution,
preferably a ready-to-use solution.
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Subject matter of the present invention is a pharmaceutical formulation of PAM
for use in therapy of a
subject according to the present invention, wherein said pharmaceutical
composition is in a freeze-dried
state.
Subject matter of the present invention is a pharmaceutical formulation for
use in therapy of a subject,
wherein said pharmaceutical formulation is administered via infusion.
Subject matter of the present invention is a pharmaceutical formulation for
use in therapy of a subject,
wherein said pharmaceutical formulation is to be administered systemically.
Therapeutic proteins for subcutaneous administration are frequently
administered at high-
concentrations. Particularly contemplated high-concentrations of therapeutic
proteins (without taking
into account the weight of chemical modifications such as PEGylation), are at
least about 70, 80, 90, 95,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 175,
180, 185, 190, 195, 200, 250,
300, 350, 400, 450, or 500 mg/ml, and/or less than about 250, 300, 350, 400,
450 or 500 mg/ml.
Exemplary high-concentrations of therapeutic proteins, such as enzymes, in the
formulation may range
from about 100 mg/ml to about 500 mg/ml. Preferably, the concentrations of the
therapeutic protein
according to the invention are in the range of about 100-300 mg/ml, more
preferred in the range of 135-
165 mg/ml, most preferred of about 150 mg/ml. A further most preferred
concentration is about 100
mg/ml. In this context a concentration of "about" a given value, e.g. the
upper or lower limit of a given
concentration range, is to be understood as encompassing all concentration
deviating up to 10% from
this given value.
Chemical modifications may be employed for protecting PAM from degradation,
extending in vivo half-
life, providing prolonged drug release, augmenting drug efficacy, while
reducing side effects, reducing
administration frequency and lowering drug dosage. Other advantages include
alleviation of pain
associated with frequent injections and significant reduction in cost of
treatment. Chemical
modifications include covalent conjugation of polymers such as PEG
(polyethyleneglycol), polysialic
acid, hyperglycosyation and mannosylation (Patel et at. 2014. Ther. Deily.
5(3): 337-365). Alternative
formulation approaches include colloidal carriers as protein delivery systems
such as microparticles,
nanoparti cl es, liposom es, carbon nanotubes and micelles (Patel etal. 2014.
Ther. Deily. 5(3): 337-365).
In one embodiment of the invention said covalently conjugated polymer is
selected from the group of
branched or unbranched polyethyleneglycol (PEG), branched or unbranclied
polypropyleneglycol
(PPG) hydroxyethyl starch (HES) or a derivative thereof, polysialic acids
(PSAs) or a derivative thereof,
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or a glycine-rich homo-amino- acid polymer (HAP) (see W02020254197 for
reference). The polymer
can be of any molecular weight.
In another aspect of the invention PAM may be administered via gene therapy.
There are two major
conventional methods of gene therapy, the first is to use viral capsids to
encapsulate a DNA sequence
of interest for introduction into mammalian tissue. Such viral capsids are
generally termed viral vectors,
and a variety of vectors have been used including HIV and adenoviruses. Such
viral vectors are generally
constructed so that they should not reproduce in-vivo, and usually contain a
reverse transcriptase that
results in splicing of the viral vector borne DNA into the host genome. The
second major conventional
method of gene therapy, DNA gene therapy, uses the much simpler method of
injection of DNA into
the patient. This introduces considerably less packaging material into the
host, and DNA constructs are
generally smaller. DNA is not incorporated into the host genome, but is
instead maintained separately
in circlets either inside the nucleus or at the nuclear wall inside the cell.
These circlets may become
associated with histones within the nucleus.
In a specific embodiment of the application, an assay is used for determining
the level of PAM and/or
its isoforms and/or fragments thereof, wherein such assay is a sandwich assay,
preferably a fully
automated assay.
In one embodiment of the application it may be a so-called POC-test (point-of-
care) that is a test
technology, which allows performing the test within less than 1 hour near the
patient without the
requirement of a fully automated assay system. One example for this technology
is the
immunochromatographic test technology.
In one embodiment of the application such an assay is a sandwich immunoassay
using any kind of
detection technology including but not restricted to enzyme label,
chemiluminescence label,
electrochemiluminescence label, preferably a fully automated assay. In one
embodiment of the invention
such an assay is an enzyme labeled sandwich assay. Examples of automated or
fully automated assay
comprise assays that may be used for one of the following systems: Roche
Elecsys , Abbott Architect ,
Siemens Centauerk, Brahms Kryptork, BiomerieuxVidask, Alere Triage , Ortho
Clinical Diagnostics
Vitros .
In a specific embodiment of the application, at least one of said two binders
is labeled in order to be
detected.
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The preferred detection methods comprise immunoassays in various formats such
as for instance
radioimmunoassay (RIA), homogeneous enzyme-multiplied immunoassays (EMIT),
chem ilum inescence- and fluorescence-immunoassays, Enzyme-linked immunoassays
(ELISA),
Luminex-based bead arrays, protein microarray assays, and rapid test formats
such as for instance
immunochromatographic strip tests.
In a preferred embodiment, said label is selected from the group comprising
chemiluminescent label,
enzyme label, fluorescence label, radioiodine label.
The assays can be homogenous or heterogeneous assays, competitive and non-
competitive assays. In
one embodiment, the assay is in the form of a sandwich assay, which is a non-
competitive immunoassay,
wherein the molecule to be detected and/or quantified is bound to a first
antibody and to a second
antibody. The first antibody may be bound to a solid phase, e.g. a bead, a
surface of a well or other
container, a chip or a strip, and the second antibody is an antibody which is
labeled, e.g. with a dye, with
a radioisotope, or a reactive or catalytically active moiety. The amount of
labeled antibody bound to the
analyte is then measured by an appropriate method. The general composition and
procedures involved
with "sandwich assays" are well-established and known to the skilled person
(The Immunoassay
Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. Way 2005); Hultschig
et at. 2006. Curr
Opin Chem Biol. 10 (1):4-10).
In another embodiment the assay comprises two capture molecules, preferably
antibodies which are
both present as dispersions in a liquid reaction mixture, wherein a first
labelling component is attached
to the first capture molecule, wherein said first labelling component is part
of a labelling system based
on fluorescence- or chemiluminescence-quenching or amplification, and a second
labelling component
of said marking system is attached to the second capture molecule, so that
upon binding of both capture
molecules to the analyte a measurable signal is generated that allows for the
detection of the formed
sandwich complexes in the solution comprising the sample.
In another embodiment, said labeling system comprises rare earth cryptates or
rare earth chelates in
combination with fluorescence dye or chemiluminescence dye, in particular a
dye of the cyanine type.
In the context of the present invention, fluorescence based assays comprise
the use of dyes, which may
for instance be selected from the group
comprising FAM (5 -or
6-carboxyfluorescein), VIC, NED, fluorescein,
fluorescein-isothiocyanate (FITC),
1RD-700/800, Cyanine dyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthen, 6-
Carboxy-2',4',7',4,7-
hexachlorofluorescein (HEX), TET, 6-Carboxy-4',5'-dichloro-2',7'-
dimethodyfluorescein (JOE),
N,N,N',N'-Tetramethy1-6-carboxyrhodamine (TAMRA),
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6-Carboxy-X-rhodamine (ROX), 5-Carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-
6G (RG6),
Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes, such as
BODIPY
TMR, Oregon Green, coumarines such as umbelliferone, benzimides, such as
Hoechst 33258;
Phenanthridines, such as Texas Red. Yakima Yellow, Alexa Fluor, PET,
ethidiumbromide, acridinium
dyes, carbazol dyes, Phenoxazine dyes, porphyrine dyes, polymethine dyes, and
the like.
In the context of the application, chemiluminescence based assays comprise the
use of dyes, based on
the physical principles described for chemiluminescent materials in (Kirk-
Othmer, Encyclopedia of
chemical technology, 4th ed. 1993. John Wiley & Sons, Vol.15: 518-562,
incorporated herein by
reference, including citations on pages 551-562). Preferred chemiluminescent
dyes arc acridinium
esters.
As mentioned herein, an "assay" or "diagnostic assay" can be of any type
applied in the field of
diagnostics. Such an assay may be based on the binding of an analyte to be
detected to one or more
capture probes with a certain affinity. Binders that may be used for
determining the level of PAM and/or
its isoforms and/or fragments thereof exhibit an affinity constant to PAM
and/or its isoforms and/or
fragments thereof of at least 107 AV, preferred 108
preferred affinity constant is greater than 109 M-
1, most preferred greater than 1010 M-1. A person skilled in the art knows
that it may be considered to
compensate lower affinity by applying a higher dose of compounds and this
measure would not lead
out-of-the-scope of the invention.
In the context of the present application, -binder molecules" are molecules
which may be used to bind
target molecules or molecules of interest, i.e. analytes (i.e. in the context
of the present invention PAM
and its isoforms and fragments thereof), from a sample. Binder molecules have
thus to be shaped
adequately, both spatially and in terms of surface features, such as surface
charge, hydrophobicity,
hydrophilicity, presence or absence of lewis donors and/or acceptors, to
specifically bind the target
molecules or molecules of interest. Hereby, the binding may for instance be
mediated by ionic, van-der-
Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions or a
combination of two or Mare of
the aforementioned interactions between the capture molecules and the target
molecules or molecules
of interest. In the context of the present invention, binder molecules may for
instance be selected from
the group comprising a nucleic acid molecule, a carbohydrate molecule, a PNA
molecule, a protein, an
antibody, a peptide or a glycoprotein. Preferably, the binder molecules are
antibodies, including
fragments thereof with sufficient affinity to a target or molecule of
interest, and including recombinant
antibodies or recombinant antibody fragments, as well as chemically and/or
biochemically modified
derivatives of said antibodies or fragments derived from the variant chain.
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In a specific embodiment said binder may be selected from the group of
antibody, antibody fragment or
non-IgG scaffold.
Chemiluminescent label may be acridinium ester label, steroid labels involving
isoluminol labels and
the like.
Enzyme labels may be lactate dehydrogenase (LDH), creatine kinase (CPK),
alkaline phosphatase,
aspartate aminotransfe rase (AST), alanine aminotransferase (ALT), acid
phosphatase, glucose-6-
phosphate dehydrogenase and so on.
In one embodiment of the application at least one of said two binders is bound
to a solid phase as
magnetic particles, and polystyrene surfaces.
Subject matter of the application is a method for determining the level of PAM
and/ or isoforms and/ or
fragments thereof in a bodily fluid sample using an assay, wherein said assay
is comprising two binders
that bind to two different epitopes of PAM, wherein the two binders are
directed to an epitope of at least
amino acids, preferably at least 4 amino acids in length.
An epitope, also known as antigenic determinant, is the part of an antigen
(e.g. peptide or protein) that
is recognized by the immune system, specifically by antibodies. For example,
the epitope is the specific
piece of the antigen to which an antibody binds. The part of an antibody that
binds to the epitope is
called a paratope. The epitopes of protein antigens are divided into two
categories: conformational
epitopes and linear epitopes, based on their structure and interaction with
the paratope.
A linear or a sequential epitope is an epitope that is recognized by
antibodies by its linear sequence of
amino acids, or primary structure and is formed by the 3-D conformation
adopted by the interaction of
contiguous amino acid residues. Conformational and linear epitopes interact
with the paratope based on
the 3-D conformation adopted by the epitope, which is determined by the
surface features of the involved
epitope residues and the shape or tertiary structure of other segments of the
antigen. A conformational
epitope is formed by the 3-D conformation adopted by the interaction of
discontiguous amino acid
residues.
In one embodiment of the application linear epitopes are related to following
sequences of immunization
peptides of PAM: peptide 1 (SEQ ID No. 11), peptide 2 (SEQ ID No. 12), peptide
(SEQ ID No. 13),
peptide 4 (SEQ ID No. 14), peptide 5 (SEQ ID No. 15), peptide 6 (SEQ ID No.
16), peptide 7 (SEQ ID
No. 17), peptide 8 (SEQ ID No. 18), peptide 9 (SEQ ID No. 19), peptide 10 (SEQ
ID No. 20), peptide
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11 (SEQ ID No. 21), peptide 12 (SEQ ID No. 22), peptide 13 (SEQ ID No. 23)
peptide 14 (SEQ ID No.
24).
In one embodiment of the application, linear and/ or conformational epitopes
are related to the following
sequences of PAM: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ
ID No. 5, SEQ
ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10.
Said epitope may comprise at least 6 amino acids, preferably at least 5 amino
acids, most preferred at
least 4 amino acids.
In one embodiment of the application said first and second binder binds to an
epitope comprised within
the following sequences of PAM: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ
ID No. 4, SEQ ID
No. 5 and SEQ ID No. 6.
In one embodiment of the application said first and second binder binds to an
epitope comprised within
the PAL subunit of PAM (SEQ ID No. 8).
In one embodiment of the application said first and second binder binds to an
epitope comprised within
the PHM subunit of PAM (SEQ ID No. 7).
In one specific embodiment of the application said first binder binds to an
epitope comprised within the
PAL subunit of PAM (SEQ ID No. 8) and said second binder binds to an epitope
comprised within the
PHM subunit of PAM (SEQ ID No. 7).
In one specific embodiment of the application said first and second binder
binds to an epitope comprised
within the following sequences of PAM: peptide 1 (SEQ ID No. 11), peptide 2
(SEQ ID No. 12), peptide
(SEQ ID No. 13), peptide 4 (SEQ ID No. 14), peptide 5 (SEQ ID No. 15), peptide
6 (SEQ ID No. 16),
peptide 7 (SEQ ID No. 17), peptide 8 (SEQ ID No. 18), peptide 9 (SEQ ID No.
19), peptide 10 (SEQ
ID No. 20), peptide 11 (SEQ ID No. 21), peptide 12 (SEQ ID No. 22), peptide 13
(SEQ ID No. 23)
peptide 14 (SEQ ID No. 24) and recombinant PAM (SEQ ID No. 10).
Use of at least two binders for the determination of the level of PAM and/ or
its isoforms and/ or
fragments thereof, wherein said at least one binder is directed to an epitope
comprised within the
following sequences of PAM: peptide 1 (SEQ ID No. 11), peptide 2 (SEQ ID No.
12), peptide (SEQ ID
No. 13), peptide 4 (SEQ ID No. 14), peptide 5 (SEQ ID No. 15), peptide 6 (SEQ
ID No. 16). peptide 7
(SEQ ID No. 17), peptide 8 (SEQ ID No. 18), peptide 9 (SEQ ID No. 19), peptide
10 (SEQ ID No. 20),
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peptide 11 (SEQ ID No. 21), peptide 12 (SEQ ID No. 22), peptide 13 (SEQ ID No.
23) peptide 14 (SEQ
ID No. 24) and recombinant PAM (SEQ ID No. 10).
Subject of the present application is a method for determining the activity of
PAM and/ or isoforms and/
or fragments thereof in a bodily fluid sample of a subject comprising the
steps
= Contacting said sample with a capture-binder that binds specifically to
active full-length PAM,
its isoforms and/ or active fragments thereof,
= Separating PAM bound to said capture-binder
= Adding a substrate of PAM to said separated PAM
= Quantifying PAM activity by measuring the conversion of the substrate of
PAM.
In a specific embodiment of the present application said method is an enzyme
capture assay (ECA, see
e.g. U55612186A, U55601986A).
In a specific embodiment of said method for determining PAM activity in a
bodily fluid sample of a
subject said separation step is a washing step that removes ingredients of the
sample that are not bound
to said capture-binder from the captured PAM and/or its isoforms and/or
fragments thereof That
separation step can be any other step that separates PAM bound to said capture-
binder from the
ingredients of said bodily fluid sample.
One embodiment of the present application involves a chemical assay for PAM.
The assay uses a peptide
substrate which reacts with PAM and/ or its isoforms and/ or fragments thereof
to form a detectable
reaction product. Alternatively, the rate of the reaction of the substrate can
be monitored to determine
the level of PAM and/or its isoforms and/or fragments thereof in a test
sample.
Assays embodying such reagents and reactions can be performed in any suitable
reaction vessel, for
example, a test tube or well of a microtiter plate. Alternatively, assay
devices may be developed in
disposable form such as dipstick or test strip device formats which are well
known to those skilled-in-
the-art and which provide ease of manufacture and use. Such disposable assay
devices may be packaged
in the form of kits containing all necessary materials, reagents and
instructions for use.
In an alternative assay embodiment, the rate at which the reaction occurs may
be detected as an
indication of the level of PAM and/ or its isoforms and/ or fragments thereof
present in the test sample.
For example, the rate at which the substrate is reacted may be used to
indicate the level of PAM and/ or
its isoforms and/ or fragments thereof present in the test sample.
Alternatively, the rate at which the
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reaction product is formed may be used to indicate the level of PAM and/ or
its isoforms and/ or
fragments thereof present in the test sample.
In yet another embodiment, a capture or binding assay may be performed to
determine the activity of
PAM and/ or its isoforms and/ or fragments thereof For example, an antibody
reactive with PAM
protein, but which does not interfere with its enzymatic activity, may be
immobilized upon a solid phase.
The test sample is passed over the immobile antibody, and PAM and/ or its
isoforms and/ or fragments
thereof, if present, binds to the antibody and is itself immobilized for
detection. A substrate may then
be added, and the reaction product may be detected to indicate the level of
PAM and/ or its isoforms
and/ or fragments thereof in the test sample. For the purposes of the present
description, the term "solid
phase" may be used to include any material or vessel in which or on which the
assay may be performed
and includes, but is not limited to, porous materials, nonporous materials,
test tubcs, wells, slides, etc.
In a specific embodiment of said method for the diagnosis or prognosis of a
disease in a subject and/or
predicting a risk of getting a disease or adverse event in a subject and/or
monitoring a disease or adverse
event in a subject by determining the level of peptidylglycine alpha-amidating
monooxygenase (PAM)
and/or its isoforms and/or fragments thereof in a sample of bodily fluid of
said subject said capture
binder is immobilized on a surface. For the determination of PAM activity, a
binder reactive with PAM
and/ or its isoforms and/ or fragments thereof, but which does not interfere
with enzymatic activity by
more than 50 %, preferably less than 40 %, preferably less than 30 %, may be
immobilized upon a solid
phase. To prevent inhibition of PAM the capture-binder should not bind PAM in
the area around the
active center and substrate binding region.
In a specific embodiment of said method for determining the level of PAM and/
or its isoforms and/ or
fragments thereof in a bodily fluid sample of a subject said binder may be
selected from the group of
antibodies, antibody fragments, non-Ig scaffolds or aptamers.
Another subject of the present application is a method for determining the
activity of PAM and/ or
isoforms and/ or fragments thereof in a bodily fluid sample of a subject
comprising the steps
= contacting said sample with a substrate (peptide-Gly) of PAM for an
interval of time at t=0 min
and t=n+1 min
= detecting the reaction product (alpha-amidated peptide) of PAM in said
sample at 1=0 mm and
t=n+1 min, and
= quantifying the activity of PAM by calculating the difference of the
reaction product between
t=0 and t=n+1.
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Another subject of the present application is a method for determining PAM
activity in a bodily fluid
sample of a subject comprising the steps
= contacting said sample with the substrate ADM-Gly of PAM for an interval
of time at t=0 min
and t=n+1 min
= detecting the reaction product ADM-NH2 of PAM in said sample at t=0 min
and t=n+1 min
using an immunoassay, and
= quantifying the activity of PAM by calculating the difference of the
reaction product ADM-NH2
between t=0 min and t=n+1 min.
The term "t=n+1 min" is a time interval, wherein n is defined as > 0 min.
One embodiment of the present application relates to a kit for performing the
method for diagnosis or
prognosis of a disease in a subject and/or predicting a risk of getting a
disease or an adverse event in a
subject and/or monitoring a disease or adverse event in a subject, wherein
said kit comprises at least two
binders directed to recombinant PAM (SEQ ID No. 10), peptide 1 (SEQ ID No.
11), peptide 2 (SEQ ID
No. 12), peptide (SEQ ID No. 13), peptide 4 (SEQ ID No. 14), peptide 5 (SEQ ID
No. 15), peptide 6
(SEQ ID No. 16), peptide 7 (SEQ ID No. 17), peptide 8 (SEQ ID No. 18), peptide
9 (SEQ ID No. 19),
peptide 10 (SEQ ID No. 20), peptide 11 (SEQ ID No. 21), peptide 12 (SEQ ID No.
22), peptide 13 (SEQ
ID No. 23) and peptide 14 (SEQ ID No. 24).
A specific embodiment of the present application relates to a kit for the
detection of the level of PAM
comprising one or more binders binding to PAM sequences selected from the
group comprising
recombinant PAM (SEQ ID No. 10), peptide 1 (SEQ ID No. 11), peptide 2 (SEQ ID
No. 12), peptide
(SEQ ID No. 13), peptide 4 (SEQ ID No. 14), peptide 5 (SEQ ID No. 15), peptide
6 (SEQ ID No. 16),
peptide 7 (SEQ ID No. 17), peptide 8 (SEQ ID No. 18), peptide 9 (SEQ ID No.
19), peptide 10 (SEQ
ID No. 20), peptide 11 (SEQ ID No. 21), peptide 12 (SEQ ID No. 22), peptide 13
(SEQ ID No. 23) and
peptide 14 (SEQ ID No. 24).
Another embodiment of the present application relates to a kit for performing
the method for
determining the activity of PAM and/ or isoforms and/ or fragments thereof in
a bodily fluid sample of
a subject, wherein said kit comprises peptide-Gly PAM as substrate, wherein
said peptide-Gly is ADM-
Gly.
The activity of PAM can be measured by detection of alpha-amidated peptides
(peptide-amide) from
their glycinated precursor peptide substrates (peptide-Gly). Nearly half of
biologically active peptides
terminate with a C-terminal alpha-amide (Vishvanatha etal. 2014.1 Biol Chem
289(18):12404-20).
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The glycinated precursor peptide substrates may be selected from the group
comprising adrenomedullin
(ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal
peptide (PAMP),
amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S,
neuromdin U,
calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid
polypeptide, chromogranin A,
insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin,
big gastrin, gastrin,
glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating
polypeptide (PACAP), secretin,
somatoliberin, peptide hi stidine methionine (PHM), vasoactive intestinal
peptide (VIP), gonadoliberin,
kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P,
neurokinin A,
neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B,
melanotropin alpha (alpha-
MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin,
vasopressin.
In a preferred embodiment said peptide-Gly is adrenomcdullin-Gly (ADM-Gly) and
said peptide-amide
is adrenomedullin-amide (ADM-NH2).
Other substrates of non-peptide character may comprise N-fatty acyl-glycines,
which are converted by
PAM to primary fatty acid amides (PFAMs) like oleamide.
In another preferred embodiment of the invention the PAM, e.g., RecPAM, is
combined the
administration of ascorbate.
Another preferred embodiment of the present application relates to a
pharmaceutical forniulation, the
formulation comprising PAM, ascorbate and/ or copper.
Another embodiment of the present application relates to a pharmaceutical
formulation, the formulation
comprising PAM in combination with ascorbate and/ or copper.
In one embodiment, the ascorbic acid compound is L-ascorbic acid or a
pharmaceutically acceptable
salt thereof; or a pharmaceutically acceptable solvate or hydrate thereof. L-
Ascorbic acid is also known
as vitamin C, L-xyloascoibic acid, 3-oxo-L-gulofuranolactone (enol form), L-3-
ketothreollexuronic acid
lactone, antiscorbutic vitamin, cevitamic acid, adenex, allercorb, ascorin,
ascorteal, ascorvit, cantan,
cantaxin, catavin C, cebicure, cebion, cecon, cegiolan, celaskon, celin,
cenetone, cereon, cergona,
cescorbat, cetamid, cetabe, cetemican, cevalin, cevatine, cevex, cevimin, ce-
vi-sol, cevitan, cevitex,
cewin, ciamin, cipca, concemin, C-vin, daviamon C. duoscorb, hybrin,
laroscorbine, lemascorb, planavit
C, proscorbin, redoxon, ribena, scorbacid, scorbu-C, testascorbic, vicelat,
vitacee, vitacimin, vitacin,
vitascorbol, and xitix.
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In one embodiment, the ascorbic acid compound is L-ascorbic acid. In another
embodiment, the ascorbic
acid compound is a pharmaceutically acceptable salt of L-ascorbic acid, or a
pharmaceutically
acceptable solvate or hydrate thereof.
Suitable bases for forming a pharmaceutically acceptable salt of L-ascorbic
acid include, but are not
limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide,
potassium hydroxide,
zinc hydroxide, and sodium hydroxide; and organic bases, such as primary,
secondary, tertiary, and
quaternary, aliphatic and aromatic amines, including, but not limited to, L-
arginine, benethamine,
benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine,
dipropylamine,
diisopropylaminc, 2-(diethylamino)-ethanol, cthanolaminc, ethylamine,
ethylonediamine,
isopropylamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine,
morpholine, 4-(2-
hydroxyethyl)-morpholine, mothylamine, piperidine, piperazine, propylaminc,
pyrrolidinc, 1-(2-
hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline,
triethanolamine,
trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-
1,3-propanediol,
and tromethamine.
In one embodiment, the ascorbic acid compound is an alkali or alkaline earth
metal salt of L-ascorbic
acid, or a pharmaceutically acceptable solvate or hydrate thereof. In another
embodiment, the ascorbic
acid compound is sodium, potassium, calcium, or magnesium L-ascorbate, or a
pharmaceutically
acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic
acid compound is sodium
L-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof In
yet another embodiment,
the ascorbic acid compound is sodium L-ascorbate, which is also known as
vitamin C sodium, ascorbin,
sodascorbate, natrascorb, cenolate, ascorbicin, or cebitate. In yet another
embodiment, the ascorbic acid
compound is potassium L-ascorbate, or a pharmaceutically acceptable solvate or
hydrate thereof. In vet
another embodiment, the ascorbic acid compound is calcium L-ascorbate, or a
pharmaceutically
acceptable solvate or hydrate thereof. In yet another embodiment, the ascorbic
acid compound is calcium
L-ascorbate. In yet another embodiment, the ascorbic acid compound is
magnesium L-ascorbate, or a
pharmaceutically acceptable solvate or hydrate thereof. In still another
embodiment, the ascorbic acid
compound is magnesium L-ascorbate.
In certain embodiments, the ascorbic acid compound is D-ascorbic acid or a
pharmaceutically acceptable
salt, or a pharmaceutically acceptable solvate or hydrate thereof
In one embodiment, the ascorbic acid compound is D-ascorbic acid. In another
embodiment, the ascorbic
acid compound is a pharmaceutically acceptable salt of D-ascorbic acid, or a
pharmaceutically
acceptable solvate or hydrate thereof
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Suitable bases for forming a pharmaceutically acceptable salt of D-ascorbic
acid include, but are not
limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide,
potassium hydroxide,
zinc hydroxide, and sodium hydroxide; and organic bases, such as primary,
secondary, tertiary, and
quaternary, aliphatic and aromatic amines, including, but not limited to, L-
arginine, benethamine,
benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine,
dipropylamine,
diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine,
ethylamine, ethyl enediamine,
isopropylamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine,
morpholine, 4-(2-
hydroxyethyp-morpholine, methylamine, piperidine, piperazine, propylamine,
pyrrolidine, 1-(2-
hydroxycthyl)-pyrrolidinc, pyridine, quinuclidinc, quinolinc, isoquinolinc,
tricthanolaminc,
trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-
1,3 -propanediol,
and tromethamine.
In one embodiment, the ascorbic acid compound is an alkali or alkaline earth
metal salt of D-ascorbic
acid, or a pharmaceutically acceptable solvate or hydrate thereof In another
embodiment, the ascorbic
acid compound is sodium, potassium, calcium, or magnesium D-ascorbate, or a
pharmaceutically
acceptable solvate or hydrate thereof In yet another embodiment, the ascorbic
acid compound is sodium
D-ascorbate, or a pharmaceutically acceptable solvate or hydrate thereof. In
yet another embodiment,
the ascorbic acid compound is sodium D-ascorbate, which is also known as
vitamin C sodium, ascorbin,
sodascorbate, natrascorb, cenolate, ascorbicin, or cebitate. In yet another
embodiment, the ascorbic acid
compound is potassium D-ascorbate, or a pharmaceutically acceptable solvate or
hydrate thereof In yet
another embodiment, the ascorbic acid compound is calcium D-ascorbate, or a
pharmaceutically
acceptable solvate or hydrate thereof In yet another embodiment, the ascorbic
acid compound is calcium
D-ascorbate. In yet another embodiment, the ascorbic acid compound is
magnesium D-ascorbate, or a
pharmaceutically acceptable solvate or hydrate thereof In still another
embodiment, the ascorbic acid
compound is magnesium D-ascorbate.
The term "solvate" refers to a complex or aggregate formed by one or More
molecules of a solute, e.g.,
a compound provided herein, and one or more molecules of a solvent, which
present in stoichiometric
or non-stoichiometric amount. Suitable solvents include, but are not limited
to, water, methanol, ethanol,
n-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent
is pharmaceutically
acceptable. In one embodiment, the complex or aggregate is in a crystalline
form. In another
embodiment, the complex or aggregate is in a non-crystalline form. Where the
solvent is water, the
solvate is a hydrate. Examples of hydrates include, but are not limited to, a
hemihydrate, monohydrate,
dihydratc, trihydratc, tctrahydratc, and pcntahydratc.
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In one embodiment, the ascorbic acid compound in each of the pharmaceutical
compositions is
independently L-ascorbic acid or a pharmaceutically acceptable salt thereof,
or a pharmaceutically
acceptable solvate or hydrate thereof. In another embodiment, the ascorbic
acid compound in each of
the pharmaceutical compositions is independently an alkali or alkaline earth
metal salt of L-ascorbic
acid, or a pharmaceutically acceptable solvate or hydrate thereof; or a
mixture thereof In yet another
embodiment, the ascorbic acid compound in each of the pharmaceutical
compositions is independently
sodium, potassium, calcium, or magnesium salt of L-ascorbic acid, or a
pharmaceutically acceptable
solvate or hydrate thereoff, or a mixture thereof In yet another embodiment,
the ascorbic acid compound
in each of the pharmaceutical compositions is independently sodium L-
ascorbate. In yet another
embodiment, the ascorbic acid compound in each of the pharmaceutical
compositions is independently
calcium L-ascorbate. In yet another embodiment, the ascorbic acid compound in
each of the
pharmaceutical compositions is independently magnesium L-ascorbatc. In still
another cmbodimcnt, the
ascorbic acid compound in each of the pharmaceutical compositions is
independently a mixture of two
or three of sodium L-ascorbate, calcium L-ascorbate, and magnesium L-
ascorbate.
With the above context, the following consecutively numbered embodiments
provide further specific
aspects of the invention:
1. Peptidylglycine alpha-amidating monooxygenase (PAM) for use as a
medicament.
2. PAM for use as a medicament for treatment of a subject, wherein said
treatment comprises:
i. reducing the potential or risk for a disease or disorder,
and/ or
reducing the occurrence of a disease or disorder, and/ or
iii. reducing the severity of a disease or disorder.
3. PAM for use as a medicament for treatment of a subject according to
embodiment 2, wherein
said disease or disorder is selected from the group comprising dementia,
cardiovascular
disorders, kidney diseases, cancer, inflammatory or infectious diseases and/or
metabolic
diseases.
4. PAM for use as a medicament for treatment of a subject according to
embodiment 2 and 3,
wherein said subject is characterized by
= a level of PAM and/or its isoforms and/or fragments thereof below a
threshold
and/ or
= a peptide-Gly/ peptide-amide ratio above a threshold
in a sample of bodily fluid of said subject.
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5. PAM for use as a medicament for treatment of a subject according to
embodiment 4, wherein
said peptide is selected from the group of comprising adrenomedullin (ADM),
adrenoinedullin-
2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin,
gastrin-
releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromdin U,
calcitonin,
calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide,
chromogranin A,
insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin,
big gastrin, gastrin,
glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating
polypeptide (PACAP),
secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive
intestinal peptide
(VIP), gonadoliberin, kisspeptin. MIF-1, metastin, neuropeptide K,
neuropeptide gamma,
substance P. neurokinin A, neurokinin B, peptide YY, pancreatic hormone,
deltorphin I, orexin
A and B, mclanotropin alpha (alpha-MSH), mclanotropin gamma, thyrotropin-
rcicasing
hormone (TRH), oxytocin, vasopressin.
6. PAM for use as a medicament for treatment of a subject according to
embodiment 4 and 5,
wherein said subject is characterized by
= an ADM-Gly/ bio-ADM ratio above a threshold and/ or
= a bio-ADM concentration below a threshold
in a bodily fluid of said patient.
7. PAM for use as a medicament for treatment of a subject according to
embodiment 3, wherein
the level of PAM and/or its isoforms and/or fragments thereof is the total
concentration of PAM
and/or its isoforms and/or fragments thereof having at least 12 amino acids or
the activity of
PAM and/or its isoforms and/or fragments thereof.
8. PAM for use as a medicament for treatment of a subject according to
embodiment 7, wherein
the total concentration of PAM and/or its isoforms and/or fragments thereof
having at least 12
amino acids or the activity of PAM and/or its isoforms and/or fragments
thereof is selected from
the group comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4,
SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.
9. PAM for use as a medicament for treatment of a subject according to
embodiments 4-8, wherein
the sample of bodily fluid of said subject is selected from the group of
blood, serum, plasma,
urine, cerebrospinal fluid (CSF), and saliva.
10. PAM for use as a medicament according to embodiment 1-9, wherein said PAM
is selected
from the group comprising isolated and/ or recombinant and/or chimeric PAM.
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11. PAM for use as a medicament according to embodiments 1-10, wherein said
recombinant PAM
is selected from the sequences comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID
No. 3, SEQ
ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID
No. 10.
12. PAM for use as a medicament for treatment of a subject according to any
embodiment 1 to 11
wherein PAM is combined with ascorbate and/ or copper.
13. Pharmaceutical formulation comprising peptidylglycine alpha-amidating
monooxygenase
(PAM).
14. Pharmaceutical formulation comprising PAM according to embodiment 13,
wherein said
pharmaceutical formulation is administered orally, epicutaneously,
subcutaneously,
intradermally, sublingually, intramuscularly, intraarterially, intravenously,
or via the central
nervous system (CNS, intracerebrally, intracerebroventricularly,
intrathecally) or via
intraperitoneal administration.
15. Pharmaceutical formulation according to embodiments 13-14, wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
16. Pharmaceutical formulation according to embodiments 13-15, wherein said
pharmaceutical
formulation is in a freeze-dried state.
17. Pharmaceutical formulation according to embodiments 13-16, wherein said
pharmaceutical
formulation is administered intra-muscular.
18. Pharmaceutical formulation according to embodiments 13-17, wherein said
pharmaceutical
formulation is administered intra-vascular.
19. Pharmaceutical formulation according to embodiment 13-18, wherein said
pharmaceutical
formulation is administered via infusion.
20. Pharmaceutical formulation according to embodiments 13-19, wherein said
pharmaceutical
formulation is to be administered systemically.
21. Pharmaceutical formulation according to embodiments 13-20, the formulation
comprising PAM
and/or optionally one or more pharmaceutically acceptable ingredients.
22. Pharmaceutical formulation according to embodiments 13-21, the formulation
comprising
PAM, ascorbate and/ or copper.
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23. Pharmaceutical formulation according to embodiments 13-22, thc formulation
comprising PAM
in combination with ascorbate and/ or copper.
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FIGURE DESCRIPTION
Fig. 1: Schematic representation of PAM Isofonii 1. Black bold arrows indicate
cleavage-sites at
double-basic amino-acids.
Fig. 2: Enzymatic reaction catalysed by PAM
Fig. 3: Representative calibration curve of recombinant PAM (ADM maturation
acitivity [AMA].
Fig. 4: Frequency distribution (histogram) of AMA in self-reported healthy
individuals (n=120)
Fig. 5: Correlation of AMA in matrix duplets (Li-heparin and scrum) from self-
reported healthy
individuals (n=20)
Fig. 6 A-L: Typical calibration curves of PAM sandwich immunoassays. A-J with
recombinant PAM
as calibration material. (A) solid phase: antibody directed to peptide 10 (SEQ
ID No. 20), tracer:
antibody directed to peptide 9 (SEQ ID No. 19); (B) solid phase: antibody
directed to peptide 10 (SEQ
ID No. 20), tracer: antibody directed to peptide 10 (SEQ ID No. 20); (C) solid
phase: antibody directed
to peptide 9 (SEQ ID No. 19), tracer: antibody directed to peptide 10 (SEQ ID
No. 20); (D) solid phase:
antibody directed to recombinant PAM (SEQ ID No. 10), tracer: antibody
directed to recombinant PAM
(SEQ ID No. 10); (E) solid phase: antibody directed to peptide 10 (SEQ ID No.
20), tracer: antibody
directed to recombinant PAM (SEQ ID No. 10); (F) solid phase: antibody
directed to peptide 13 (SEQ
ID No. 23), tracer: antibody directed to peptide 10 (SEQ ID No. 20); (G) solid
phase: antibody directed
to peptide 14 (SEQ ID No. 24), tracer: antibody directed to peptide 13 (SEQ ID
No. 23); (H) solid phase:
antibody directed to recombinant PAM (SEQ ID No. 10), tracer: antibody
directed to peptide 13 (SEQ
ID No. 23); (I) solid phase: antibody directed to peptide 13 (SEQ ID No. 23),
tracer: antibody directed
to peptide 9 (SEQ ID No. 19); (J) solid phase: antibody directed to peptide 10
(SEQ ID No. 20), tracer:
antibody directed to peptide 13 (SEQ ID No. 23). K and L with native PAM (EDTA-
Plasina) as
calibration material: (K) solid phase: antibody directed to peptide 14 (SEQ ID
No. 24), tracer: antibody
directed to peptide 13 (SEQ ID No. 23); (L) solid phase: antibody directed to
peptide 10 (SEQ ID No.
20), tracer: antibody directed to peptide 13 (SEQ ID No. 23).
Fig. 6 M-0: Enzyme capture assay (ECA) - (M) solid phase antibody directed to
peptide 10 (SEQ ID
No. 20); (N) solid phase antibody directed to full-length PAM (SEQ ID No. 10);
(0) solid phase
antibodies directed against peptide 7 (SEQ ID No. 17), peptide 8 (SEQ ID No.
18), peptide 9 (SEQ ID
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No. 19), peptide 13 (SEQ ID No. 23) and peptide 14 (SEQ ID No. 24) with
recombinant PAM/ heparin
plasma used as sample.
Fig. 7: Typical ADM-Gly dose/ signal curve
Fig. 8: ADM maturation activity (PAM activity) in MPP-study (prediction of
Alzheimer's disease)
Fig. 9: Kaplan-Meier-Plot (prediction of Alzheimer's disease [AD] in MPP-
study)
Fig. 10: ADM maturation activity (PAM activity) in MPP-study (prediction of
colorectal cancer [CRC])
Fig. 11: MR-proADM in MPP-study (prediction of colorectal cancer [CRC])
Fig. 12: Kaplan-Meier-Plot (prediction of colorectal cancer [CRC] in 1VIPP-
study)
Fig. 13: Kaplan-Meier-Plot (prediction of heart failure in MPP-study)
Fig. 14: Kaplan-Meier-Plot (prediction of atrial fibrillation in MPP-study)
Fig. 15: ADM maturation activity (PAM activity) for diagnosis of prevalent
Alzheimer's disease (AD)
Fig. 16: Formation of bio-ADM by native plasma PAM with and without exogenous
ADM-Gly as
substrate
Fig. 17: Formation of bio-ADM from native plasma ADM-Gly by native plasma PAM
and effect of
exogenous recombinant PAM
Fig. 18: Shift of ADM-Gly/bio-ADM ratio due to the reaction of native plasma
PAM and the effect of
exogenous recombinant PAM on the ADM-Gly/bio-ADM ratio
Fig. 19: ADM maturation activity (PAM activity) in rat-plasma prior to and
after application of
recombinant human PAM or placebo
Fig. 20: One-phase decay fit of ADM maturation activity (PAM activity) in rat
plasma after application
of recombinant human PAM
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Fig. 21
Intravenous injections of placebo (open circles), PAM (closed squares),
ascorbate (open triangles) and
a combination of both (open squares) in rats. The effect of injected compounds
was tested in vitro: (A)
PAM-AMA assayed as described in example 3 in absence of exogenous ascorbate.
(B) Effect on
circulating bio-ADM levels after injection of the compounds. Bio-ADM levels
were normalized to
levels of Placebo (set as 100%) for each time-point. Significance was tested
in comparison to placebo
using the two-way ANOVA model with Dunnet's correction. *: p=0.042; **: p=
0.0036 ¨ 0.0073; ***:
p=0.0003; ****: p=0.0001; n.s. : not significant.
Fig. 22: Half-life of recombinant PAM in rats, determined from one-phase decay
model.
Fig. 23: Amidating activity in healthy human volunteers before (Oh) and after
oral ascorbate uptake
measured without addition of exogenous ascorbate. AMA at t = Oh was set as
100%.
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EXAMPLES
Example 1 ¨ Production of recombinant PAM
PAM cDNA was synthesized according to Uniprot Accession No. P19021 encoding
amino acids 21-834
of the PAM protein involving codon optimization for expression in mammalian
cells. The signal
sequence of PAM was replaced with human serum albumin signal sequence
(MKWVTFISLLFLFSSAYSFR [SEQ ID No. 9]). At the C-terminus of PAM a hexa-
histidine tag was
added linked via a GS linker to PAM. The sequence of recombinant PAM (amino
acids 21-834 of PAM
without signal sequence and hexa-histidine tag) is shown in SEQ ID No. 10. The
cDNA was cloned into
an expression vector (plasmid DNA) using a 5'-NotI and a 3' HindIII
restriction site. The expression
vector harboring the cDNA for PAM expression was replicated in- and prepared
from E. coli. as a low-
endotoxin preparation.
HEK-INV cells were transfected with the expression vector using INVect
transfection reagents in serum
free suspension culture. The transfection rate was controlled via co-
transfection with a GFP- (green
fluorescent protein) containing expression vector. Cultivation of cells was
carried out in presence of
valproic acid and Penicillin-Streptomycin at 37 C and 5% CO2. Cells were
harvested via centrifugation
when viability reached <60% (>2000g, 30-45 mM, 2-8 C). Cell culture
supernatant (CCS) was washed
times with 100 mM Tris/HC1, pH 8.0 via tangential flow filtration (TFF, 30 kDa
cut-off).
Purification of recombinant PAM included application of buffer exchanged CCS
on a Q-sepharose fast
flow resin (GE Healthcare) with a NaCl gradient (up to 2 M) elution. Amidating
activity containing
fractions were pooled and applied onto a Superdex 200pg (GE Healthcare) size
exclusion
chromatography column with a 100 mM Tris/HC1, 200 mM NaCl, pH8.0 elution
buffer. Amidating
activity containing fractions were pooled, dialyzed against 100 mM Tris HC1,
200 mM NaCl, pH 8.0,
sterile filtered (0.2 um). Endotoxin load was determined by Charles River PTS
Endosafe system and
was below 5 EU/mL.
Example 2 ¨ Production of antibodies
Anti-PAM antibodies according to the present invention may be synthesised as
follows:
PAM peptides for immunization were synthesized, see Table 1, (Peptides &
Elephants, Hennigsdorf,
Germany) with an additional C-terminal cysteine (if no cysteine is present
within the selected PAM-
sequence) residue for conjugation of the peptides to Bovine Serum Albumin
(BSA). The peptides were
covalently linked to BSA by using Sulfolink-coupling gel (Perbio-science,
Bonn, Germany). The
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coupling procedure was performed according to the manual of Perbio.
Recombinant PAM was produced
by InVivo Biotech Services, Hennigsdorf, as described in example 1.
Table 1: PAM immunization peptides
Name (amino acid position*) Sequence
Peptide 1 (aa 42-56) (SEQ ID No. 11) CLGTTRPVVPIDSSD
Peptide 2 (aa 109-128) (SEQ ID No. 12) CNMPSSTGSYWFCDEGTCTD
Peptide 3 (aa 168-180) (SEQ ID No. 13) YGDISAFRDNNKD
Peptide 4 (aa 204-216) (SEQ ID No. 14) SVDTVIPAGEKVV
Peptide 5 (aa 329-342) (SEQ ID No. 15) CTQNVAPDMFRTIP
Peptide 6 (aa 291-310) (SEQ ID No. 16) TGEGRTEATHIGGTSSDEMC
Peptide 7 (aa 234-244) (SEQ ID No. 17) YRVHTHHLGKV
Peptide 8 (aa 261-276) (SEQ ID No. 18) QSPQLPQAFYPVGHPV
Peptide 9 (aa 530-557) (SEQ ID No. 19) RGDHVWDGNSFDSKFVYQQIGLGPIEED
Peptide 10 (aa 611-631) (SEQ ID No. 20) EGPVLILGRSMQPGSDQNHFC
Peptide 11 (aa 562-579 (SEQ ID No. 21) IDPNNAAVLQSSGKNLFY
Peptide 12 (aa 745-758) (SEQ ID No. 22) NGKPHFGDQEPVQG
Peptide 13 (aa 669-687) (SEQ ID No. 23) WGEESSGSSPLPGQFTVPH
Peptide 14 (aa 710-725) (SEQ ID No. 24) CFKTDTKEFVREIKHS
Recombinant PAM SEQ ID No. 10
* according to SEQ ID No. 1; amino acid (aa)
Balb/c mice were intraperitoneally (i.p.) injected with 100 jig recombinant
PAM or
100 pg PAM-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold
Adjuvant), 100 jag and
100 pg at day 14 (emulsified in complete Freund's adjuvant) and 50 pg and 50
pg at day 21 and 28 (in
incomplete Freund's adjuvant). The animal received an intravenous (i.v.)
injection of 50 pg recombinant
PAM at day 40 or 50 pg PAM-peptide-BSA-conjugates dissolved in saline at day
45. Three days later
the mice were sacrificed and the immune cell fusion was performed.
Splenocytes from the immunized mice and cells of the myeloma cell line SP2/0
were fused with 1 ml
50% polyethylene glycol for 30 s at 37 C. After washing, the cells were seeded
in 96-well cell culture
plates. Hybrid clones were selected by growing in HAT medium (RPMI 1640
culture medium
supplemented with 20% fetal calf serum and HAT-Supplement). After one week,
the HAT medium was
replaced with HT Medium for three passages followed by returning to the normal
cell culture medium.
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The cell culture supernatants were primarily screened for recombinant PAM
binding IgG antibodies two
weeks after fusion. Therefore, recombinant PAM (SEQ ID No. 10) was immobilized
in 96-well plates
(100 ng/ well) and incubated with 50 vti cell culture supernatant per well for
2 hours at room temperature.
After washing of the plate, 50 IA/ well POD-rabbit anti mouse IgG was added
and incubated for 1 h at
RT.
After a next washing step, 50 p.1 of a chromogen solution (3.7 mM o-phenylene-
diamine in
citrate/hydrogen phosphate buffer, 0.012 % 1+02) were added to each well,
incubated for 15 minutes at
RT and the chromogenic reaction stopped by the addition of 50 Ill 4N sulfuric
acid. Absorption was
detected at 490 mm.
The positive tested microcultures were transferred into 24-well plates for
propagation. After retesting
the selected cultures were cloned and re-cloned using the limiting-dilution
technique and the isotypes
were determined.
Antibodies raised against recombinant human PAM or PAM-peptides were produced
via standard
antibody production methods (Marx et al. 1997) and purified via Protein A. The
antibody purities were
> 90 % based on SDS gel electrophoresis analysis.
Example 3 - PAM activity assay
Human serum or Li-Heparin plasma from self-reported healthy volunteers was
used as source of human
native PAM. Each sample (201i1) was diluted two-fold in 100 mM Tris-HC1 in
duplicate. The amidation
reaction was initiated by addition of 160 jil of PAM-reaction buffer (100 mM
Tris-HC1, pH 7.5, 6.25
iiM CuSO4, 2.5 mM L-ascorbate, 125 ittg/mL catalase, 62.5 iiM amastatin,
250iiM leupeptin, 36 ng/mL
synthetic ADM-Gly and 375 lig/mL NT-ADM antibody). Afterwards, 100 1 of each
individual reaction
of duplicated samples were combined and transferred into 20 pi of 200 mM EDTA
to terminate the
amidation reaction and to generate t=0 minutes reaction time-point followed by
incubation at 37 C for
40 minutes. Afterwards the non-terminated reactions were stopped with 101.1.1
of 200 mM EDTA. To
determine the PAM activity, bio-ADM as reaction product was quantified in each
sample using the
sphingotestO bio-ADM immunoassay (Weber etal. 2017). The amidation assay was
calibrated using a
6-point calibration curve generated with human recombinant PAM of known
activity. Samples and
calibrators were treated in the same manner. Relative light units (RLU t40min-
t0min) determined via
sphingotestO bio-ADM immunoassay for each sample were fitted against the RLU
(t40min-t0min) of
the calibrator to determine the PAM activity in the samples. PAM activity is
described as
-adrenomedullin maturation activity" (AMA) in lig bio-ADM formed per hour and
L of sample.
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A typical PAM calibration curve is shown in figure 3. The distribution of AMA
in Li-Heparin samples
from n=120 self-reported healthy volunteers are shown in figure 4. The median
PQR] of Li-Heparin
AMA was 18.4 jtg/(L*11) [13.5-21.9]. The 10th and 90th percentile was 10.5 and
24.2 jig/(L*11),
respectively. The 25th, 97.5t11 and 99=---,th
percentile was 8.1, 31.6 and 40.8 jig/(L*h). In addition, matched
serum samples from n=20 subjects were measured and revealed a highly
significant correlation
(r = 0.89; p <0.0001) (Figure 5), although AMA values in serum were
approximately 40% lower when
compared to Li-Heparin.
Example 4 ¨ PAM immunoassays
Antibodies against recombinant PAM (SEQ ID No. 10) and PAM peptides (SEQ ID
No. 11 to 24) were
raised as described in example 1.
The technology used was a sandwich luminescence immunoassay, based on
Akridinium ester labelling.
4.1. Labelled compound (tracer)
Purified antibodies (0.2 g/L) were labelled by incubation in 10% labelling
buffer (500 mmol/L sodium
phosphate, pH 8.0) with 1:5 mol/L ratio of MACN-acridinium-NHS-ester (1 g/L,
InVent GmbH) for 20
min at 22 'C. After adding 5% 1 mol/L Tris-HC1, pH 8.0, for 10 min, the
respective antibody was
separated from free label via CentriPure P10 columns (emp Biotech GmbH). The
purified labelled
antibody was diluted in 300 mmo1/1 potassium phosphate, 100 mmo1/1 NaC1, 10
mmo1/1 Na-EDTA, 5
g/1 Bovine Serum Albumin (pH 7.0). The final concentration was approximately
20 ng of labelled
antibody per 150 jiL.
4.2. Solid phase
White polystyrene microtiter plates (Greiner Bio-One International AG) were
coated (18 h at 20 C)
with the respective antibody (2 jig/0.2 mL per well 50 mmol/L Tris-HC1, 100
mmol/L NaC1, pH 7.8).
After blocking with 30 g/L Karion, 5 g/L BSA (protease free), 6.5 mmol/L
monopotassium phosphate,
3.5 mmol/L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum-dried.
4.3 Calibration
The assay was calibrated, using dilutions of recombinant PAM as described in
Example 1. The typical
concentration range was within of 5 ¨ 5,000 ng/mL.
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4.4. PAM immunoassays:
4.4.1. PAM-LIA
One-Step version: 50 [EL of samples /calibrators were pipetted into pre-coated
microtiter plates. After
adding 200 p.L of labelled antibody in buffer (300 mmol/L potassium phosphate,
100 mmol/L NaC1, 10
mmol/L Na-EDTA, 50 umol/L amastatin, 100 umol/L leupeptin, 0.1% bovine IgG,
0.02% mouse IgG,
0.5% BSA, pH 7.0), the microtiter plates were incubated for 20 h at 2-8 C
under agitation at 600 rpm.
Unbound tracer was removed by washing 5 times (each 350 L per well) with
washing solution (20
mmol/L PBS, 1 g/L Triton X-100, pH 7.4). Wellbound chemiluminescence was
measured for 1 s per
well by using the Centro LB 960 microtiter plate luminescence reader (Berthold
Technologies).
Two-Step version: 50 itiL of samples /calibrators were pipetted into pre-
coated microtiter plates. After
adding 200 iaL of buffer (as described in one-step version), the microtiter
plates were incubated for 15-
20 h at 2-8 'V under agitation at 600 rpm. Unbound sample was removed by
washing 4 times (each 350
vt.1_, per well) with washing solution with subsequent addition of 200111 of
tracer material and incubation
of microtiter plates at room temperature for 2h. Unbound tracer was removed by
washing 4 times (each
350 1AL per well) with washing solution. Wellbound chemiluminescence was
measured for 1 s per well
by using the Centro LB 960 microtiter plate luminescence reader (Berthold
Technologies).
Results: Antibodies bound to the solid phase and labelled antibodies directed
to the different PAM
immunization peptides as well as full-length (recombinant) PAM (see example 2)
were tested with
recombinant PAM as well as blood samples. Exemplary standard curves for
different antibody
combinations are shown in figure 6 (A-L). Figures 6 (A-J) with recombinant PAM
as calibration
material: (A) solid phase: antibody directed to peptide 10 (SEQ ID No. 20),
tracer: antibody directed to
peptide 9 (SEQ ID No. 19); (B) solid phase: antibody directed to peptide 10
(SEQ ID No. 20), tracer:
antibody directed to peptide 10 (SEQ ID No. 20); (C) solid phase: antibody
directed to peptide 9 (SEQ
ID No. 19), tracer: antibody directed to peptide 10 (SEQ ID No. 20); (D) solid
phase: antibody directed
to recombinant PAM (SEQ ID No. 10), tracer: antibody directed to recombinant
PAM (SEQ ID No.
10), (E) solid phase. antibody directed to peptide 10 (SEQ ID No. 20), tracer.
antibody directed to
recombinant PAM (SEQ ID No. 10); (F) solid phase: antibody directed to peptide
13 (SEQ ID No. 23),
tracer: antibody directed to peptide 10 (SEQ ID No. 20); (G) solid phase:
antibody directed to peptide
14 (SEQ ID No. 24), tracer: antibody directed to peptide 13 (SEQ ID No. 23);
(H) solid phase: antibody
directed to recombinant PAM (SEQ ID No. 10), tracer: antibody directed to
peptide 13 (SEQ ID No.
23); (I) solid phase: antibody directed to peptide 13 (SEQ ID No. 23), tracer:
antibody directed to peptide
9 (SEQ ID No. 19); (J) solid phase: antibody directed to peptide 10 (SEQ ID
No. 20), tracer: antibody
directed to peptide 13 (SEQ ID No. 23). Figures 6 (K and L) with native PAM
(EDTA-Plasma) as
calibration material: (K) solid phase: antibody directed to peptide 14 (SEQ ID
No. 24), tracer: antibody
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directed to peptide 13 (SEQ ID No. 23); (L) solid phase: antibody directed to
peptide 10 (SEQ ID No.
20), tracer: antibody directed to peptide 13 (SEQ ID No. 23). With all
antibody combinations PAM was
also detectable in human plasma and serum samples.
4.4.2. Enzyme capture Assay (ECA) for the detection of PAM activity
Enzyme capture assays were established to detect the activity of PAM. 50 [tt
of samples /calibrators
were pipetted into pre-coated microtiter plates (as described in 4.2.). After
adding 200 u1_, of buffer (300
mmol/L potassium phosphate, 100 mmol/L NaCl, 50 mon amastatin, 100 mon
leupeptin, 0.1%
bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0) the microtiter plates were
incubated for 1 h at room
temperature under agitation at 600 rpm. Unbound sample was removed by washing
4 times (each 350
uL per well) with washing solution with subsequent addition of 200 tl rcaction
buffer per well and
incubation at 37 C. Reaction buffer including all components and final
concentrations were as described
in Example 3, with the exceptions that 100 g/mL NT-ADM-antibody and 288 ng/mL
ADM-Gly were
used. Reaction was terminated at several time-points by transferring 10iil of
each individual reaction
into 1900 of EDTA containing buffer (300 mmol/L potassium phosphate, 100
mmol/L NaC1, 10
mmol/L Na-EDTA, 50 mon amastatin, 100 mon leupeptin, 0.1% bovine IgG, 0.02%
mouse IgG,
0.5% BSA, pH 7.0). Terminated reactions were applied onto the sphingotestk bio-
ADM immunoassay
for quantification of produced bio-ADM. A typical standard curve using an
antibody directed to PAM
immunization peptide 10 (SEQ ID No. 20) as solid phase is shown in figure 6 M.
Fig 6 N shows a typical standard curve using an antibody directed to full-
length recombinant PAM (SEQ
ID No. 10). Further antibodies directed to peptide 7 (SEQ ID No. 17), peptide
8 (SEQ ID No. 18),
peptide 9 (SEQ ID No. 19), peptide 13 (SEQ ID No. 23) and peptide 14 (SEQ ID
No. 24) were used as
solid phase for the enzyme capture assay and a sample (2501.11) of recombinant
PAM or heparin plasma
was measured for PAM activity (Fig. 6 0). These results show, that the
antibodies can be used to detect
PAM activity in human samples using the ECA technique. PAM was also detectable
in plasma and
serum samples.
In a further step, PAM activity (as described in example 3) and PAM
concentration using a PAM-LIA
(solid phase antibody directed against full-length PAM, tracer antibody
directed against peptide 13 [SEQ
ID No. 23]) were determined in heparin samples from healthy volunteers (n=26).
PAM activity and
PAM concentration correlated significantly as shown in Figure 6 P (Spearman
r=0.49, p=0.0109).
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Example 5 ¨ ADM-Gly immunoassay
ADM-Gly was quantified as based on Weber et al. (Weber et at. 2017. JA1M 2(2):
222-233) for
bioactive ADM with the following modifications: the tracer-antibody used for
ADM-Gly detection,
labelled with MACN-acridinium-NHS, was directed to the C-terminal glycine of
ADM-Gly. The assay
was calibrated with synthetic ADM-Gly. The limit of detection (LOD) was 10
pg/mL of ADM-Gly.
Cross-reactivity of antibody directed to the C-terminal glycine of ADM with
bio-ADM was in the range
between 6 and 50 % in a concentration dependent manner. All determined ADM-Gly
concentrations
were corrected for cross-reactivity as follows: For each ADM-Gly
quantification additional
quantification of bio-ADM in corresponding samples was performed using the
sphingotestO bio-ADM
immunoassay. The corresponding bio-ADM values were used to determine the
signal (RLU) generated
with the antibody directed to C-terminal glycine of ADM on a bio-ADM
calibration curve. The
determined signal (RLU) was used to calculate the false-positive ADM-Gly
concentration (pg/mL)
using the ADM-Gly calibration curve. This concentration was subtracted from
the initially determined
ADM-Gly concentration. A typical standard curve is shown in Figure 7.
Example 6 ¨ Prediction of diseases in healthy subjects
The Malmo Preventive Project (MPP) was funded in the mid-1970s to explore CV
risk factors in general
population, and enrolled 33,346 individuals living in Malmo (Fea'orawski et
at. 2010. Eur Heart J 31:
85 91). Between 2002 and 2006, a total of 18,240 original participants
responded to the invitation
(participation rate, 70.5%) and were screened including a comprehensive
physical examination and
collection of blood samples (Fava et at. 2013. Hypertension 2013: 61: 319-26).
The re-examination in
MPP is in the present study regarded as the baseline. Subjects with prior CVD
at baseline were excluded.
An informed consent was obtained from all participants and the Ethical
Committee of Lund University,
Lund, Sweden, approved the study protocol.
A commercial fully automated homogeneous time-resolved fluoro-immunoassay was
used to measure
MR-proADM in plasma (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf,
Germany) (Caruhel et at. 2009. Clin Biochem. 42 (7-8):725-8).
Bio-ADM was measured as described by Weber et al. 2017 (Weber etal. 2017.
JA1VIA 2(2): 222-233).
AMA was determined in 4942 serum samples from MPP as described in example 3.
Each sample was
measured in duplicate. Samples, controls and calibrators were treated in the
same manner. Baseline
clinical characteristics of AMA after stratification to Quartiles is shown in
table 2.
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Table 2: Baseline clinical characteristics according to quartile (Q) of AMA at
baseline of subjects
analysed
Q1 Q2 Q3 Q4
(n=1235) (n=1236) (n=1236) (n=1235)
AMA in i.tg/(L*h) (SD) 9.416 11.66 (0.46) 13.39 (0.57) 17(3)
N/A
(1.21)
AMA range 3.8 - 10.86 10.86 - 12.47 - 14.47-
72.15 N/A
12.47 14.47
Age in years (SD) 68.97 69.16 (6.28) 69.34 (6.38) 70.45
(6.07) <
(6.18)
0.0001
Current smoking, n (%) 188 (15.2) 217 (17.6) 255 (20.6) 287
(23.2)
0.0001
Systolic blood pressure in 144 145.1 144.8 147.6 (21.34)
<
mmHg (SD) (19.77) (19.83) (20.33)
0.0002
Diastolic blood pressure 82.83 84.04 83.12 84.45 (11.51)
0.0041
mmHg (SD) (10.12) (10.83) (10.61)
Diabetes Mellitus, n (%) 166 (13.4) 127 (10.3) 113 (9.1)
127 (10.3) 0.0043
Glucose in mmol/L (SD) 6.024 5.78 (1.21) 5.794 (1.37) 5.753
(1.28) 0.0299
(1.95)
N/A: not applicable
Statistical analysis: Values are expressed as means and standard deviations,
medians and interquartile
ranges (IQR), or counts and percentages as appropriate. Group comparisons of
continuous variables
were performed using the Kruskal-Wallis test. Biomarker data were log-
transformed. Cox proportional-
hazards regression was used to analyze the effect of risk factors on survival
in uni- and multivariable
analyses. The assumptions of proportional hazard were tested for all
variables. For continuous variables,
hazard ratios (HR) were standardized to describe the HR for a biomarker change
of one IQR. 95%
confidence intervals (CI) for risk factors and significance levels for chi-
square (Wald test) are given.
The predictive value of each model was assessed by the model likelihood ratio
chi-square statistic. The
concordance index (C index) is given as an effect measure. It is equivalent to
the concept of AUC
adopted for binary outcome. For multivariable models, a bootstrap corrected
version of the C index is
given. Survival curves plotted by the Kaplan-Meier method were used for
illustrative purposes. To test
for independence of PAM from clinical variables we used the likelihood ratio
chi-square test for nested
models. All statistical tests were 2-tailed and a two-sided p-value of 0.05
was considered for
significance.
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6.2. Prediction of Alzheimer's disease
3954 samples with information about dementia diagnosis were selected (n=174
with incident AD).
Information about dementia diagnoses was requested from the Swedish National
Patient Register
(SNPR). The diagnoses in the register were collected according to different
revisions of the International
Classification of Diseases (ICD) codes 290, 293 (ICD-8), 290, 331 (ICD-9) or
FOO, F01, F03, G30 (ICD-
10). Since 1987, SNPR includes all in-patient care in Sweden and, in addition,
contains data on
outpatient visits including day surgery and psychiatric care from both private
and public caregivers
recorded after 2000. All-cause dementia was diagnosed according to the
criteria of the Diagnostic and
Statistical Manual of Mental Disorders (DSM)-III revised edition, whilst the
DSM-IV criteria were
applied for the Alzheimer's disease and vascular dementia diagnoses. Diagnoses
were validated by a
thorough review of medical records as well as ncuroimaging data when
available. A research physician
assigned the final diagnosis for each patient and a geriatrician specialized
in cognitive disorders was
consulted in unresolved cases. The PAM activity (AMA) was determined as
described in example 3.
AMA in the MPP cohort is shown in figure 8: AMA in patients developing AD over
time (incident AD,
n=174) are significantly lower compared to the non-AD group (p=0.01).
Reduced serum AMA strongly predicts Alzheimer's disease with a Hazard Ratio
(HR) of 0.74 (CI 0.6
¨ 0.88; p<0.001) and a HR of 0.72 (CI 0.6 ¨ 0.85) when adjusted for age (table
3). Figure 9 shows a
Kaplan-Meier Plot for the prediction of Alzheimer's disease using AMA
(prevalent AD cases were
excluded from the analysis). The lowest tertile is associated with the highest
risk of getting AD.
Furthermore, AMA as a predictor of AD was independent from bio-ADM
concentrations. Both markers
contribute to AD prediction. While the C-Index for AMA alone is 0.571 (CI
0.525 ¨ 0.616; Chi2 10.97)
the C-index for both combined markers, i.e., AMA and bio-ADM is 0.595 (Chi2
18.96; p<0.0001).
Moreover, AMA in combination with bio-ADM and MR-proADM concentrations further
improve the
prediction of incident Alzheimer. While MR-proADM alone had no predictive
value for AD, the
combination of AMA, bio-ADM and MR-proADM showed a C-index of 0.622 (Chi'
26.73, p-0.00001).
Table 3: Prediction of Alzheimer's disease
Biomarker Hazard Ratio (HR) p-Value C-Index (CI) Chi2
(Cl)
AMA 0.72 (0.6-0.85) p<0.001 0.571 (0.525-
10.97
0.616)
AMA, bio-ADM p<0.0001 0.595 18.96
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6.3. Prediction of colorectal cancer (CRC)
AMA of subjects with and without incident CRC is shown in figure 10. The AMA
in patients developing
CRC over time (n=93) are significantly lower compared to the non-CRC group
(p=0.0008; Kruskal-
Wallis). In contrast, the MR-proADM concentrations in patients developing CRC
over time arc higher
compared to the non-CRC group (p=0.023) as shown in figure 11.
Results for the single markers and marker combinations are shown in Table 4.
Reduced serum AMA
(age-adjusted) strongly predicts development of CRC with a Hazard Ratio (HR)
of 0.68 (p<0.0001).
Figure 12 shows a Kaplan-Meier Plot for the prediction of CRC with AMA
(prevalent cases were
excluded from the analysis). The lowest tertile is associated with the highest
risk of CRC development
(p<0.005).
Increased MR-proADM concentrations predict development of CRC with a HR of
1.36 p<0.05). The
highest quartile is associated with the highest risk of CRC development
(p=0.051).
While bio-ADM concentrations were not predictive for development of CRC, a
combination of bio-
ADM and AMA showed an improved CRC prediction (see table 4). In addition, a
combination of AMA
and MR-proADM further improved the prediction of CRC development.
In summary, reduced AMA values predict development of CRC. Increased MR-proADM
concentrations
also predict development of CRC. A combination of AMA with bio-ADM or MR-
proADM enhances
the predictive value for CRC.
Table 4: Prediction of colorectal cancer
Biomarker Hazard Ratio (HR) p-Value C-Index (CI)
Chi'
(CI)
AMA 0.68 (0.6-0.85) p<0.00001
0.588 (0.535-0.641) 8.51
AMA, bioADM p<0.002 0.598
12.48
MR-proADM 1.36 (1.08-1.72) p<0.05 0.587 (0.532-
0.642) 6.27
AMA, MR-proADM p<0.0005 0.612
16.51
6.5. Prediction of cardiovascular disorders
Cardiovascular disorder analyses were performed in 4942 samples with
information about death- and
cardiovascular events from the MPP cohort. Information about cardiovascular
events and diagnoses was
requested from the Swedish National Patient Register (SNPR). The diagnoses in
the register were
collected according to different revisions of the International Classification
of Diseases (ICD) codes.
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Since 1987, SNPR includes all in-patient care in Sweden and, in addition,
contains data on outpatient
visits including day surgery and psychiatric care from both private and public
caregivers recorded after
2000. The PAM activity (AMA) was determined as described in example 3. Within
of the total set of
4942 serum samples from the MPP study cohort 278 subjects developed heart
failure (incident heart
failure) and 633 subjects developed atrial fibrillation (incident atrial
fibrillation) during follow-up period
of 12.8 years.
Elevated serum AMA strongly predicts incident heart failure (83 prevalent HF
cases were excluded
from the analyses) with a Hazard Ratio (HR) of 1.537 (CI 1.169 ¨ 2.021;
p<0.0007) (Table 5). Figure
13 shows a Kaplan-Meier Plot for the prediction of heart failure using AMA.
High AMA is associated
with increased risk of getting heart failure.
Elevated serum AMA strongly predicts incident atrial fibrillation (267
prevalent AF cases were
excluded from the analyses) with a Hazard Ratio (HR) of 1.459 (Cl 1.214 ¨
1.752; p<0.0001) (Table 5).
Figure 14 shows a Kaplan-Meier Plot for the prediction of atrial fibrillation
using AMA. High AMA is
associated with increased risk of getting atrial fibrillation.
Table 5: Prediction of cardiovascular disorders
Q1 Q2
(n=3707) (n=1119)
Heart failure
Number of Events 186 92
Logrank Hazard Ratio 1.537
(1.169 ¨ 2.021)
(95% CI)
Chi2 (ref)
11.56
p-value =0.0007
Atrial fibrillation
Q1 Q2
(n=3534) (n=1141)
Number of Events 436 197
Logrank Hazard Ratio 1.459
(1.214 ¨ 1.752)
Chi2 (ref)
19.59
p-value <0.0001
Example 7 ¨ Diagnosis of diseases
7.1. Diagnosis of Alzheimer's disease
Serum samples from 27 individuals with diagnosed Alzheimer's disease were
obtained from InVent
Diagnostica GmbH. The AD diagnosis is based on cognitive tests (CERAD, DemTec,
MMST and
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Clock-Drawing test) as well as on MRI (Magnetic resonance imaging) and CT-
scans. As controls, 67
serum samples from self-reported healthy volunteers were used. AMA was
detected as described in
example 3.
As shown in figure 15 patients from the AD-cohort showed significantly lower
serum AMA when
compared to the control-cohort (n=67; p<0.0001).
7.2. Diagnosis of cardiovascular and metabolic disorders
In the total set of 4942 scrum samples from the MPP study cohort, 267 cases of
prevalent atrial
fibrillation, 83 cases of prevalent chronic heart failure and 533 cases of
prevalent diabetes were present.
Significant elevation of scrum AMA (p<0.0001) was observed in prevalent atrial
fibrillation (mean
AMA: 13.92 AMA-Units, n=267) when compared to individuals free of prevalent
atrial fibrillation
(mean AMA: 12.8 AMA-Units, n=4675). Significant elevation of serum AMA
(p=0.0019) was observed
in prevalent chronic heart failure (mean AMA: 14.31 AMA-Units, n=83) when
compared to individuals
free of prevalent heart failure (mean AMA: 12.84 AMA-Units, n=4859).
Significant reduction of serum
AMA (p=0.0035) was observed in prevalent diabetes (mean AMA: 12.69 AMA-Units,
n= 533) when
compared to individuals free of prevalent diabetes (mean AMA: 12.89 AMA-Units.
n=4409).
Example 8 ¨ Conversion of ADM-Gly to bio-ADM by native and recombinant PAM
a) Conversion of ADM-Gly to bio-ADM by native PAM
Human Li-Heparin plasma (pool of 3 specimen with low ADM-Gly (<50 pg/mL)) was
used as source
of human native PAM. The amidation reaction was performed in a total volume of
120 tl at 37 C. 96
IA of plasma were spiked with ADM-Gly (5 ng/mL final concentration). As
control, equal volumes of
100 mM Tris-HC1, pH 7.5 were added to untreated plasma. The prepared samples
were allowed to chill
for 15 minutes at room temperature. The amidation reaction was started by
addition of 24 of PAM-
reaction buffer resulting in final concentrations of 2 mM L-ascorbate and 5
viM CuSO4, respectively,
with a final concentration for ADM-Gly 4 ng/mL. After 0 min, 30 min, 60 min
and 90 min of incubation
at 37 C, the reaction was stopped by addition of Na-EDTA (20mM final
concentration). The
concentration of bio-ADM in the reaction sample was quantified using the
sphingotestO bio-ADM
immunoassay as described recently (Weber et al 2017 ,IAL11/12(2)- 222-233). No
change in bio-ADM
concentration was detected in low ADM-Gly samples without addition of
exogenous ADM-Gly. When
ADM-Gly was added to the sample, a linear formation of bio-ADM was detected
within 90 minutes
(Figure 16).
b) Conversion of ADM-Gly to bio-ADM by exogenous (recombinant) PAM
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In a further experiment we investigated the formation of bio-ADM from
endogenous ADM-Gly by
native human plasma PAM and the effect of addition of exogenous recombinant
human PAM. Human
Li-Heparin plasma (pool of 5 specimen with high ADM-Gly (> 400 pg/mL)) was
used as source of
human native PAM and human native ADM-Gly. The amidation reaction was
performed in a total
volume of 315 at 37 C. 250 IA of plasma were spiked with 65 td
reaction buffer with or without
recombinant PAM (see Example 1) to initiate the amidation reaction. Final
concentrations in the reaction
samples were: 2 mM L-ascorbate, 5 ttM CuSO4, 50 viM Amastatin, 200 !LIM
Leupeptin and 100 lag/mL
Catalase. The concentration of exogenous recombinant PAM was 500 ng/ml. After
0 min, 30 min, 55
min and 80 min of incubation at 37 C, the reaction was stopped by addition of
Na-EDTA (20 mM final
concentration). The concentration of bio-ADM in the reaction sample was
quantified using the
sphingotest bio-ADM immunoassay as described recently (Weber et al. 2017.
JAL11/I 2(2): 222-233).
The concentrations of ADM-Gly were determined as described in example 5.
As shown in Figure 17, endogenous human PAM enzyme is capable of the
conversion of endogenous
human ADM-Gly to bioADM in a time-dependent manner. Moreover, ADM-Gly/bio-ADM
ratio is
shifted towards bio-ADM in a time dependent manner (Figure 18). These data
clearly indicate that the
PAM exerts its function of c-terminal amidation of peptide-hormones not only
in the lumen of secretory
vesicles but also in the circulation. An approximate doubling of the PAM
concentration in the reaction
sample by addition of exogenous recombinant human PAM leads to an increase of
the bio-ADM
synthesis rate from endogenous human ADM-Gly (Figure 17) by an averaged factor
of 1.8 at each time-
point. Furthermore, the ADM-Gly consumption is increased with a faster shift
of the ADM-Gly/bio-
ADM ratio towards bio-ADM (Figure 18). These in vitro findings show an
unexpectedly high potential
of recombinant human PAM in shifting potentially unfavourable circulating ADM-
Gly/bio-ADM ratios
towards bio-ADM.
Example 9 ¨Application of recombinant PAM (in vivo half-life of PAM in rats)
Two animals (Wistar-rat, male, 2-3 month of age) received 17 lag of
recombinant human PAM (see
example 1) as a single dose in a total volume of 500 p.1 (in phosphate-
buffered saline). One
control-animal received 500 1 of PBS. Blood sampling (Li-Heparin Plasma) was
carried out 30 min
prior to application and 30 min, 2h, 4h, 8h, 24h and 48h, respectively, after
application.
AMA in plasma samples was determined as described in example 3. AMA prior to
application was
defined as 100% and AMA after application was normalized to AMA prior to
application. AMA (%)
from the three animals is shown in Figure 19. Half-Life of the PAM enzyme was
determined from the
averaged activities of the Enzyme group animals (Figure 20) using a one phase
decay fit (Graph Pad
Prism). The half-life of the PAM Enzyme was 47 min.
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In a second experiment three animals (Wistar-rat, male, 2-3 month of age)
received 25 lag of
recombinant human PAM (see example 3) as a single dose in a total volume of
500 vtl. One
control-animal received 5041 of PBS. Blood sampling (Li-Heparin Plasma) was
carried out 15 minutes
prior to application and 15, 30, 45, 60, 90 min as well as 2h, 3h, 4h, and 8h,
respectively, after
application. Half-life of the PAM enzyme was determined as described above and
was with 60 min
comparable to the determination described above.
These data clearly demonstrate the capability of intravenously administered
recombinant human PAM
to convert circulating ADM-Gly into bio-ADM in vivo. The administration of
recombinant human PAM
led to a bio-ADM increase with a maximum after 4h with a half-life of the PAM
enzyme of
approximately 53.5 min.
Example 10 ¨ Injection of recombinant PAM in combination with ascorbate in
rats
Endotoxin-free, recombinant PAM (SinoBiological) was buffer exchanged into
sterile phosphate buffer
saline (PBS, Dulbecco) and adjusted to 50 [tg/mL. Sterile ascorbate solution
for injection (200 mg/mL,
vitamin C 1000, WORWAG Pharma) was purchased in a local pharmacy and adjusted
under sterile
conditions with PBS to 40 mg/mL. For combined injections of PAM and ascorbate,
the compounds were
prepared separately in equal volumes (1001,1g/mL or 80 mg/mL for PAM and
ascorbate, respectively).
Both compounds were combined directly prior to injection to result in
concentrations of 50 g/mL for
PAM and 40 mg/mL for ascorbate. Sterile PBS was used as placebo. All samples
were stored at -80 C
until use. Animals, male Wistar rats, 2-3 month of age, were assigned into 4
groups (placebo, ascorbate,
PAM, PAM + ascorbate) with 3 animals per group. Each animal in the individual
group received 500
of the respective compound intravenously as a single dose injection. Blood
sampling was performed
as Li-Heparin 30 min prior to and 15, 30, 45, 60, 120, 180, 240 and 300 min
post injection. PAM
activities were determined as described in example 3.
Injection of Ascorbate in rats resulted in non-significant elevation of
endogenous PAM activity, when
measured without exogenous Ascorbate addition in assay (Figure 21A, open
triangles). PAM Activity
remained elevated for 60 minutes after injection with its maximal elevation
after 15 min past injection.
After 120 min past injection measured activity was comparable to PAM activity
in the placebo group.
Injection of recombinant human PAM in rats resulted in highly significant
elevation of circulating PAM
activity, when measured without exogenous Ascorbate addition (Figure 21A,
filled squares). Maximal
elevation of PAM activity by the factor of 6 when compared to placebo group
was reached 15 minutes
past injection. Circulating PAM activity remained significantly elevated for
60 minutes after injection
(30 min, 45 min and 60 mm) with a decreasing tendency in this time period.
After 120 min past injection
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measured PAM activity was not significantly different to PAM activity in the
placebo group. Injection
of a combination of recombinant human PAM and Ascorbate in rats resulted in
highly significant
elevation of circulating PAM activity, when measured without exogenous
Ascorbate addition (Figure
21A, open squares). Maximal elevation of PAM activity by the factor of 24 when
compared to placebo
group was reached 15 minutes past injection. Circulating PAM activity remained
significantly elevated
for 60 minutes after injection (30 min, 45 mm, 60 min) with a decreasing
tendency in this time period.
After 120 min past injection measured activity remained significantly elevated
when compared to PAM
activity in the placebo group. Application of enzyme- and ascorbate-free
placebo did not increase the
circulating PAM Activity (Figure 21A, open circles).
The determination of bio-ADM in circulation of all treatment-groups
surprisingly revealed elevation of
circulating bio-ADM concentration in a time-dependent manncr:
After application of Ascorbate, bio-ADM concentration was elevated (non-
significant) by the factor of
1.4 after 15 min and after 30 mm, by the factor of 1.2 after 45 min and
returned to baseline levels after
60 minutes (Figure 21B, filled circles). After application of recombinant
human PAM (Figure 21B, open
squares), bio-ADM concentration was highly significantly elevated by the
factor of 1.8 after 15 min and
by the factor of 2.4 after 30 min. After 45 mm bio-ADM concentrations
decreased but remained
significantly elevated by the factor of 2. After 60 min bio-ADM concentrations
were not significantly
different when compared to the placebo group. Highest elevation of circulating
bio-ADM was observed
in the PAM+Ascorbate combination group (Figure 21B, filled squares): After
application of
recombinant human PAM and Ascorbate, bio-ADM concentration was highly
significantly elevated by
the factor of 2.3 after 15 min and by the factor of 3.2 after 30 min. After 45
min bio-ADM concentrations
decreased but remained significantly elevated by the factor of 2.8. After 60
min bio-ADM
concentrations remained elevated by the factor of 1.6, but the difference was
not significant when
compared the placebo group. In all 3 groups (Ascorbate, PAM and PAM+Ascorbate)
bio-ADM
concentrations were not significantly different to the Placebo group after 120
minutes. Application of
enzyme- and Ascorbate-free placebo did not increase the bio-ADM concentration
in plasma (Figure
21B, open circles). Bio-ADM concentrations of the placebo group were set as
100% for each timepoint
and bio-ADM concentrations in the treatment groups were normalized to the
placebo group.
These data clearly demonstrate the capability of intravenously administered
recombinant human PAM
and a combination of recombinant human PAM with Ascorbate to convert
circulating ADM-Gly into
bio-ADM in vivo. The administration of recombinant human PAM led to a bio-ADM
increase with a
maximum after 30 minutes.
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As shown in Figure 22, the half-life of recombinant human PAM in rats is 52.7
minutes. The determined
half-life is comparable to the half-life determined in example 9 (Figure 20),
while the precision of the
half-life determination was increased by generation of additional datapoints
between 0 and 60 minutes
after injection
Example 11 - Effect of ascorbate on circulating human PAM activity
Healthy volunteers (n=4) received 2000 mg of vitamin C (Dr. Scheffler,
Additivak Vitamin C) as an
oral single dose. Blood sampling was performed prior to- and 1, 2 and 3 hours
post administration. Basal
amidating activity was determined from Li-heparin plasma and serum as
described in example 3 in
absence of exogenous ascorbate addition and is shown in Figure 23.
Surprisingly, PAM activity determined without exogenous ascorbate addition was
significantly elevated
lh after oral Ascorbate uptake by the factor of 1.7 when compared to PAM
activity prior to ascorbate
uptake (Figure 23). Further, after 2h and 3h post oral ascorbate uptake, PAM
activity remained elevated
by the factor of ¨2. These data clearly demonstrate that oral Ascorbate uptake
is suitable for modulation
of circulating PAM activity in humans.
SEQUENCES
SEQ ID NO: 1 - Prepro-PAM isoform 1 AS 1-973
10 20 30 40 50
MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV
60 70 80 90 100
PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT
110 120 130 140 150
VITHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG
160 170 180 190 200
FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY
210 220 230 240 250
LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV
260 270 280 290 300
RNGQWTL1GR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH
310 320 330 340 350
IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP
360 370 380 390 400
VKSDMVMMHE FIHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE
410 420 430 440 450
REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN
460 470 480 490 500
AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE
510 520 530 540 550
ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG
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560 570 580 590 600
LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH
610 620 630 640 650
QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG
660 670 680 690 700
YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV
710 720 730 740 750
ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF
760 770 780 790 800
GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT
810 820 830 840 850
NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM
860 870 880 890 900
QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGDSEH
910 920 930 940 950
KLETSSGRVL GRFRGKGSGG LNLGNFFASR KGYSRKGFDR LSTEGSDQEK
960 970
EDDGSESEEE YSAPLPALAP SSS
SEQ ID NO: 2 - Prepro-PAM isoform 2 AS 1-868
10 20 30 40 50
MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV
60 70 80 90 100
P1DSSDFALD 1RMPGVTPKQ SDTYFCMSMRIPVDEEAFV1 DFKPRASMDT
110 120 130 140 150
VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG
160 170 180 190 200
FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY
210 220 230 240 250
LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV
260 270 280 290 300
RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH
310 320 330 340 350
IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP
360 370 380 390 400
VKSDMVMMHE HEIKETEYKDK IPLLQQPKRE EEEVLDQDFH MEEALDWPGV
410 420 430 440 450
YLLPGQVSGV ALDPKNNLVI FHRGDHVWDG NSFDSKFVYQ QIGLGPIEED
460 470 480 490 500
TILVIDPNNA AVLQSSGKNL FYLPHGLSID KDGNYWVTDV ALHQVFKLDP
510 520 530 540 550
NNKEGPVLIL GRSMQPGSDQ NHFCQPTDVA VDPGTGAIYV SDGYCNSRIV
560 570 580 590 600
QFSPSGKFIT QWGEESSGSS PLPGQFTVPH SLALVPLLGQ LCVADRENGR
610 620 630 640 650
IQCFKTDTKE FVREIKHSSF GRNVFAISYI PGLLFAVNGK PHFGDQEPVQ
660 670 680 690 700
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GFVMNFSNGE IIDIFKPVRK HFDMPHDIVA SEDGTVYIGD AHTNTVWKFT
710 720 730 740 750
LTEKLEHRSV KKAGIEVQEI KEAEAVVETK MENKPTSSEL QKMQEKQKLI
760 770 780 790 800
KEPGSGVPVV LITTLLVIPV VVLLAIAIFI RWKKSRAFGD SEHKLETSSG
810 820 830 840 850
RVI,GRERGKG SGGINI,GNEF ASRKGYSRKG FDRT,STEGSD QEKEDDGSES
860
EEEYSAPLPA LAPSSS
SEQ ID No.: 3 - Prepro-PAM isoform 3 AS (amino acids 829-896 of SEQ ID No. I
missing)
20 30 40 50
MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV
60 70 80 90 100
PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT
110 120 130 140 150
VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG
160 170 180 190 200
FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY
210 220 230 240 250
LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV
260 270 280 290 300
RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEA'TH
310 320 330 340 350
IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP
360 370 380 390 400
VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE
410 420 430 440 450
REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN
460 470 480 490 500
AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE
510 520 530 540 550
ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG
560 570 580 590 600
LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYVVVTDVALH
610 620 630 640 650
QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGATYVSDG
660 670 680 690 700
YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV
710 720 730 740 750
ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF
760 770 780 790 800
GDQEPVQGFV MNFSNGETID IFKPVRKHFD MPHDIVASED GTVYIGDAHT
810 820 830 840 850
NTVWKFTLTE KLEHRSVKKA GIEVQEIKDS EHKLETSSGR VLGRFRGKGS
860 870 880 890 900
GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL
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APSSS
SEQ ID No. 4 - Prepro-PAM isoform 4 (amino acids 829-914 of SEQ ID No. 1
missing)
10 20 30 40 50
MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV
60 70 80 90 100
PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT
110 120 130 140 150
VHFIMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG
160 170 180 190 200
FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY
210 220 230 240 250
LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVHTHH LGKVVSGYRV
260 270 280 290 300
RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH
310 320 330 340 350
IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP
360 370 380 390 400
VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE
410 420 430 440 450
REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN
460 470 480 490 500
AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE
510 520 530 540 550
ALDWPGVYLL PGQVSGVALD PKNNLVIFHR GDHVWDGNSF DSKFVYQQIG
560 570 580 590 600
LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH
610 620 630 640 650
QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG
660 670 680 690 700
YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV
710 720 730 740 750
ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF
760 770 780 790 800
GDQEPVQGFV MNFSNGEIID IFKPVRKHFD MPHDIVASED GTVYIGDAHT
810 820 830 840 850
NTVWKFTLTE KLEHRSVKKA GIEVQEIKGK GSGGLNLGNF FASRKGYSRK
860 870 880
GFDRLSTEGS DQEKEDDGSE SEEEYSAPLP ALAPSSS
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SEQ ID No. 5 - Prepro-PAM Isoform 5 (Isoform 1 with an additional an in
position 896)
10 20 30 40 50
MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV
60 70 80 90 100
PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT
110 120 130 140 150
VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG
160 170 180 190 200
FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY
210 220 230 240 250
LMMSVD'TVIP AGEKVVNSDI SCHYKNYPMH VFAYRVH'TIT-1LGKVVSGYRV
260 270 280 290 300
RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH
310 320 330 340 350
IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP
360 370 380 390 400
VKSDMVMMHE HHKETEYKDK 1PLLQQPKRE EEEVLDQGDF YSLLSKLLGE
410 420 430 440 450
REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN
460 470 480 490 500
AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE
510 520 530 540 550
ALDWPGVYLL PGQVSGVALD PKNNLV1FHR GDHVWDGNSF DSKFVYQQIG
560 570 580 590 600
LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH
610 620 630 640 650
QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGA1YVSDG
660 670 680 690 700
YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV
710 720 730 740 750
ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF
760 770 780 790 800
GDQEPVQGFV MNFSNGEIID 1FKPVRKHFD MPHD1VASED GTVY1GDAHT
810 820 830 840 850
NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM
860 870 880 890 900
QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGADSE
910 920 930 940 950
HKLETSSGRV LGRFRGKGSG GLNLGNFFAS RKGYSRKGFD RLSTEGSDQE
960 970
KEDDGSESEE EYSAPLPALA PSSS
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SEQ ID No. 6 - Prepro-PAM Isoform 6 (amino acids 897-914 of SEQ ID No. 1
missing)
10 20 30 40 50
MAGRVPSLLV LLVFPSSCLA FRSPLSVFKR FKETTRPFSN ECLGTTRPVV
60 70 80 90 100
PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR IPVDEEAFVI DFKPRASMDT
110 120 130 140 150
VHHMLLFGCN MPSSTGSYWF CDEGTCTDKA NILYAWARNA PPTRLPKGVG
160 170 180 190 200
FRVGGETGSK YFVLQVHYGD ISAFRDNNKD CSGVSLHLTR LPQPLIAGMY
210 220 230 240 250
LMMSVDTVIP AGEKVVNSDI SCHYKNYPMH VFAYRVH'TIT-1LGKVVSGYRV
260 270 280 290 300
RNGQWTLIGR QSPQLPQAFY PVGHPVDVSF GDLLAARCVF TGEGRTEATH
310 320 330 340 350
IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT QNVAPDMFRT IPPEANIPIP
360 370 380 390 400
VKSDMVMMHE HHKETEYKDK IPLLQQPKRE EEEVLDQGDF YSLLSKLLGE
410 420 430 440 450
REDVVHVHKY NPTEKAESES DLVAEIANVV QKKDLGRSDA REGAEHERGN
460 470 480 490 500
AILVRDRIHK FHRLVSTLRP PESRVFSLQQ PPPGEGTWEP EHTGDFHMEE
510 520 530 540 550
ALDWPGVYLL PGQVSGVALD PKNNLVIEHR GDHVWDGNSF DSKFVYQQIG
560 570 580 590 600
LGPIEEDTIL VIDPNNAAVL QSSGKNLFYL PHGLSIDKDG NYWVTDVALH
610 620 630 640 650
QVFKLDPNNK EGPVLILGRS MQPGSDQNHF CQPTDVAVDP GTGAIYVSDG
660 670 680 690 700
YCNSRIVQFS PSGKFITQWG EESSGSSPLP GQFTVPHSLA LVPLLGQLCV
710 720 730 740 750
ADRENGRIQC FKTDTKEFVR EIKHSSFGRN VFAISYIPGL LFAVNGKPHF
760 770 780 790 800
GDQEPVQGFV MNFSNGEIID IFKPVRKHED MPHDIVASED GTVY1GDAHT
810 820 830 840 850
NTVWKFTLTE KLEHRSVKKA GIEVQEIKEA EAVVETKMEN KPTSSELQKM
860 870 880 890 900
QEKQKLIKEP GSGVPVVLIT TLLVIPVVVL LAIAIFIRWK KSRAFGGKGS
910 920 930 940 950
GGLNLGNFFA SRKGYSRKGF DRLSTEGSDQ EKEDDGSESE EEYSAPLPAL
APSSS
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SEQ ID No. 7 - PHM subunit of PAM
10 20 30 40 50
FKETTRPF SN ECLGTTRPVV PIDSSDFALD IRMPGVTPKQ SDTYFCMSMR
60 70 80 90 100
IPVDEEAFVI DFKPRA S MD T VHEIMLLFGCN MPS STGSYWF CDEGTCTDKA
110 120 130 140 150
NILYAWARNA PPTRLPKGVG FRVGGETGSK YFVLQVHYGD I S AFRDNNKD
160 170 180 190 200
C SGV SLHLTR LPQPLIAGMY LMMSVDTVIP AGEKVVN S DI SCHYKNYPMH
210 220 230 240 250
VF AYRVHTHH LGKVVSGYRV RNGQWTLIGR Q SP Q LP Q A FY PVGHPVDV SF
260 270 280 290 300
GDLLAARCVF TGEGRTEATH IGGTSSDEMC NLYIMYYMEA KHAVSFMTCT
310 320 330 340 350
QNVAPDMFRT IPPEANIPIP VKSDMVMMHE HHKETEYKDK IPLLQQPKRE
360 370 380 390 400
EEEVLDQGDF Y SLLSKLLGE RED V VHVHKY N PTEKAE SE S DLVAEIAN V V
410 420 430 440 450
QKKDLGRSDA REGAEHERGN AILVRDRIHK FHRLVSTLRP PE SRVF SLQQ
460
PPPGEGTWEP EHTG
SEQ ID No. 8 - PAL subunit of PAM
10 20 30 40 Sc)
DFHMEEALDW PGVYLLPGQV SGVALDPKNN LVIFHRGDHV WDGNSFDSKF
60 70 80 90 100
VYQQIGLGPI EEDTILVIDP NNA AVLQ S SG KNLFYLPHGL SIDKDGNYWV
110 120 130 140 150
TDVALHQVFK LDPNNKEGPV LILGRSMQPG SD QNHFCQPT DVAVDPGTGA
160 170 180 190 200
IYV SDGYCNS RIVQF SP SGK FITQWGEES S GS
SPLPGQFT VPHSLALVPL
210 220 230 240 250
LGQLCVADRE NGRIQ CFKTD TKEFVREIKH
SSFGRNVFAI SYIPGLLFAV
260 270 280 290 300
NGKPHFGDQE PVQGFVMNF S NGEIIDIFKP VRKHFDMPHD IVA S EDGTVY
310 320
IGDAHTNTVW KFTLTEKLEH RSV
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SEQ ID No. 9 - signal sequence human serum albumin
10 20
MKWVTFISLL FLESSAYSFR
SEQ ID No. 10 - Sequence of recombinant human PAM
10 20 30 40 50
SPLSVFKRFK ETTRPFSNEC LGTIRPV VPI DSSDFALDIR MPGVTPKQSD
60 70 80 90 100
TYFCMSMRIP VDEEAFVIDF KPRASMDTVH HMLLFGCNMP SSTGSYWFCD
110 120 130 140 150
EGTCTDKANI LYAWARNAPP TRLPKGVGFR VGGETGSKYF VLQVHYGDIS
160 170 180 190 200
AFRDNNKDCS GVSLHLTRLP QPLIAGMYLM MSVDTVIPAG EKVVNSDISC
210 220 230 240 250
HYKNYPMHVF AYRVHTHHLG KVVSGYRVRN GQWTLIGRQS PQLPQAFYPV
260 270 280 290 300
GHPVDVSFGD LLAARCVFTG EGRTEATHIG GTSSDEMCNL YIMYYMEAKH
310 320 330 340 350
AVSFMTCTQN VAPDMFRTIP PEANIPIPVK SDMVMMHEHH KETEYKDKIP
360 370 380 390 400
LLQQPKREEE EVLDQGDFYS LLSKLLGERE DVVHVHKYNP TEKAESESDL
410 420 430 440 450
VAHAN V V QK KDLGRSDARE GAEHERGNAI LVRDRIHKFH RLVSTLRPPE
460 470 480 490 500
SRVFSLQQPP PGEGTWEPEH TGDFHMEEAL DWPGVYLLPG QVSGVALDPK
510 520 530 540 550
NNLVIFHRGD HVWDGNSFDS KFVYQQIGLG PIEEDTILVI DPNNAAVLQS
560 570 580 590 600
SGKNLFYLPH GLSIDKDGNY WVTDVALHQV FKLDPNNKEG PVLILGRSMQ
610 620 630 640 650
PGSDQNHFCQ PTDVAVDPGT GAIYVSDGYC NSRIVQFSPS GKFITQWGEE
660 670 680 690 700
SSGSSPLPGQ F'TVPHSLALV PLLGQLCVAD RENGRIQCFK TDTKEFVREI
710 720 730 740 750
KHSSFGRNVF AISYIPGLLF AVNGKPFIFGD QEPVQGFVMN FSNGEIIDIF
760 770 780 790 800
KPVRKHFDMP HDIVASEDGT VYIGDAHTNT VWKFTLTEKL EHRSVKKAGI
810
EV QEIKEAEA V VGS
CA 03168769 2022- 8- 19
WO 2021/170816 PCT/EP2021/054869
SEQ ID No. 11 - Peptide 1 (aa 42-56 of PAM SEQ ID No. 1)
CLGTTRPVVP ID S SD
SEQ ID No. 12 - Peptide 2 (aa 109-128 of PAM SEQ ID No. 1)
CNMPSSTGSY WFCDEGTCTD
SEQ ID No. 13 - Peptide 3 (aa 168-180 of PAM SEQ ID No. 1)
YGDISAFRDN NKD
SEQ ID No. 14 - Peptide 4 (aa 204-216 of PAM SEQ ID No. 1)
SVDTVIPAGE KVV
SEQ ID No. 15 - Peptide 5 (aa 329-342 of PAM SEQ ID No. 1)
CTQNVAPDMF RTIP
SEQ ID No. 16- Peptide 6 (aa 291-310 of PAM SEQ ID No. 1)
10 20
TGEGR TEA TH IGGTS SDEMC
SEQ ID No. 17 - Peptide 7 (aa 234-244 of PAM SEQ ID No. 1)
YRVHTHHLGK V
SEQ ID No. 18 - Peptide 8 (aa 261-276 of PAM SEQ ID No. 1)
Q SPQLP Q A FY PVGHPV
SEQ ID No. 19 - Peptide 9 (aa 530-557 of PAM SEQ ID No. 1)
10 20
RGDHVWDGNS FDSKEVYQQ1 GLGP1EED
SEQ ID No. 20 - Peptide 10 (aa 611-631 of PAM SEQ ID No. 1)
10 20
EGPVLILGRS MQPGSDQNI-IF C
SEQ ID No. 21 - Peptide 11 (aa 562-579 of PAM SEQ ID No. 1)
IDPNNAAVLQ SSGKNLFY
SEQ ID No. 22 - Peptide 12 (aa 745-758 of PAM SEQ ID No. 1)
NGKPHFGDQE PVQG
CA 03168769 2022- 8- 19
WO 2021/170816
PCT/EP2021/054869
66
SEQ ID No. 23 - Peptide 13 (aa 669-687 of PAM SEQ ID No. 1)
WGEES SGS SP LPGQFTVPH
SEQ ID No. 24 - Peptide 14 (aa 710-725 of PAM SEQ ID No. 1)
CEKTDTKEFV REIKHS
CA 03168769 2022- 8- 19
